Label-free methods for performing assays using a colorimetric resonant reflectance optical biosensor

ABSTRACT

Methods are provided for detecting biomolecular interactions. The use of labels is not required and the methods can be performed in a high-throughput manner. The invention also relates to optical devices.

PRIORITY

This application is a continuation-in-part of U.S. application Ser. No.10/227,908, entitled “Amine Chemical Surface Activation Process And TestMethod For A Plastic Colorimetric Resonant Biosensor,” filed Aug. 26,2002, and U.S. application Ser. No. 10/180,374, entitled “ColorimetricResonant Biosensor Microarray Readout Instrument,” filed Jun. 26, 2002,and U.S. application Ser. No. 10/180,647, entitled “ColorimetricResonant Biosensor Microtiter Plate Readout Instrument” filed Jun. 26,2002, which are continuations-in-part of U.S. application Ser. No.10/059,060, filed Jan. 28, 2002 and U.S. application Ser. No.10/058,626, filed Jan. 28, 2002 now U.S. Pat. No. 6,951,715, which arecontinuations-in-part of U.S. application Ser. No. 09/930,352, filedAug. 15, 2001, which claims the benefit of U.S. provisional applicationNo. 60/244,312 filed Oct. 30, 2000; U.S. provisional application No.60/283,314 filed Apr. 12, 2001; and U.S. provisional application No.60/303,028 filed Jul. 3, 2001, all of which are incorporated herein intheir entirety.

TECHNICAL AREA OF THE INVENTION

The invention relates to methods for detecting biomolecularinteractions. The detection can occur without the use of labels and canbe done in a high-throughput manner. The invention also relates tooptical devices.

BACKGROUND OF THE INVENTION

With the completion of the sequencing of the human genome, one of thenext grand challenges of molecular biology will be to understand how themany protein targets encoded by DNA interact with other proteins, smallmolecule pharmaceutical candidates, and a large host of enzymes andinhibitors. See e.g., Pandey & Mann, “Proteomics to study genes andgenomes,” Nature, 405, p. 837–846, 2000; Leigh Anderson et al.,“Proteomics: applications in basic and applied biology,” Current Opinionin Biotechnology, 11, p. 408–412, 2000; Patterson, “Proteomics: theindustrialization of protein chemistry,” Current Opinion inBiotechnology, 11, p. 413–418, 2000; MacBeath & Schreiber, “PrintingProteins as Microarrays for High-Throughput Function Determination,”Science, 289, p. 1760–1763, 2000; De Wildt et al., “Antibody arrays forhigh-throughput screening of antibody-antigen interactions,” NatureBiotechnology, 18, p. 989–994, 2000. To this end, tools that have theability to simultaneously quantify many different biomolecularinteractions with high sensitivity will find application inpharmaceutical discovery, proteomics, and diagnostics. Further, forthese tools to find widespread use, they must be simple to use,inexpensive to own and operate, and applicable to a wide range ofanalytes that can include, for example, polynucleotides, peptides, smallproteins, antibodies, and even entire cells.

Biosensors have been developed to detect a variety of biomolecularcomplexes including oligonucleotides, antibody-antigen interactions,hormone-receptor interactions, and enzyme-substrate interactions. Ingeneral, biosensors consist of two components: a highly specificrecognition element and a transducer that converts the molecularrecognition event into a quantifiable signal. Signal transduction hasbeen accomplished by many methods, including fluorescence,interferometry (Jenison et al., “Interference-based detection of nucleicacid targets on optically coated silicon,” Nature Biotechnology, 19, p.62–65; Lin et al, “A porous silicon-based optical interferometricbiosensor,” Science, 278, p. 840–843, 1997), and gravimetry (A.Cunningham, Bioanalytical Sensors, John Wiley & Sons (1998)).

Of the optically-based transduction methods, direct methods that do notrequire labeling of analytes with fluorescent compounds are of interestdue to the relative assay simplicity and ability to study theinteraction of small molecules and proteins that are not readilylabeled. Direct optical methods include surface plasmon resonance (SPR)(Jordan & Corn, “Surface Plasmon Resonance Imaging Measurements ofElectrostatic Biopolymer Adsorption onto Chemically Modified GoldSurfaces,” Anal. Chem., 69:1449–1456 (1997)), grating couplers (Morhardet al., “Immobilization of antibodies in micropatterns for celldetection by optical diffraction,” Sensors and Actuators B, 70, p.232–242, 2000), ellipsometry (Jin et al., “A biosensor concept based onimaging ellipsometry for visualization of biomolecular interactions,”Analytical Biochemistry, 232, p. 69–72, 1995), evanascent wave devices(Huber et al., “Direct optical immunosensing (sensitivity andselectivity),” Sensors and Actuators B, 6, p. 122–126, 1992), andreflectometry (Brecht & Gauglitz, “Optical probes and transducers,”Biosensors and Bioelectronics, 10, p. 923–936, 1995). Theoreticallypredicted detection limits of these detection methods have beendetermined and experimentally confirmed to be feasible down todiagnostically relevant concentration ranges. However, to date, thesemethods have yet to yield commercially available high-throughputinstruments that can perform high sensitivity assays without any type oflabel in a format that is readily compatible with the microtiterplate-based or microarray-based infrastructure that is most often usedfor high-throughput biomolecular interaction analysis. Therefore, thereis a need in the art for methods that can achieve these goals.

SUMMARY OF THE INVENTION

The invention provides methods for detecting binding or cleavage of oneor more specific binding substances to the colorimetric resonantreflectance optical biosensor surface, or to their respective bindingpartners which are immobilized on the surface of a colorimetric resonantreflectance optical biosensor. This and other embodiments of theinvention are provided by one or more of the embodiments describedbelow.

One embodiment of the invention provides a method of detecting cleavageof one or more entire specific binding substances from a surface of acolorimetric resonant reflectance optical biosensor, wherein one or morespecific binding substances are immobilized on the surface of thebiosensor at distinct locations. The method comprises detecting acolorimetric resonant reflectance optical biosensor peak wavelengthvalue (PWV) of the distinct locations; applying one or more cleavingmolecules to the distinct locations; detecting colorimetric resonantreflectance optical PWVs of the distinct locations; and comparing theinitial PWVs above with the subsequent PWVs above. The cleavage of oneor more entire specific binding substances is detected, and a peakwavelength value (PWV) is a relative measure of the specific bindingsubstance that is bound to the biosensor.

A cleaving molecule is a molecule that can cleave another molecule. Forexample, a cleaving molecule can be an enzyme, including proteases,lipases, nucleases, lyases, peptidases, hydrolases, ligases, kinases andphosphatases. A colorimetric resonant reflectance optical biosensor cancomprise an internal surface of a microtiter well, a microtiter plate, atest tube, a petri dish or a microfluidic channel. One or more specificbinding substances can be immobilized onto the surface of the biosensorvia a nickel group, amine group, an aldehyde group, an acid group, analkane group, an alkene group, an alkyne group, an aromatic group, analcohol group, an ether group, a ketone group, an ester group, an amidegroup, an amino acid group, a nitro group, a nitrile group, acarbohydrate group, a thiol group, an organic phosphate group, a lipidgroup, a phospholipid group or a steroid group. The specific bindingsubstance can be immobilized on the surface of the colorimetric resonantreflectance optical biosensor via physical adsorption, chemical binding,electrochemical binding, electrostatic binding, hydrophobic binding orhydrophilic binding.

One or more specific binding substances can be arranged in an array ofdistinct locations on the surface of a biosensor, wherein the distinctlocations define one or more array spots of, for example, about 50–500microns, or about 150–200 microns in diameter. A specific bindingsubstance can be selected from the group consisting of nucleic acids,peptides, protein solutions, peptide solutions, single or doublestranded DNA solutions, RNA solutions, RNA-DNA hybrid solutions,solutions containing compounds from a combinatorial chemical library,antigens, polyclonal antibodies, monoclonal antibodies, single chainantibodies (scFv), F(ab) fragments, F(ab′)2 fragments, Fv fragments,small organic molecules, cells, viruses, bacteria or biological samples.A biological sample can be selected from the group consisting of blood,plasma, serum, gastrointestinal secretions, homogenates of tissues ortumors, synovial fluid, feces, saliva, sputum, cyst fluid, amnioticfluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen,lymphatic fluid, tears and prostatic fluid.

The method of detecting cleavage of one or more entire specific bindingsubstances from a surface of a colorimetric resonant reflectance opticalbiosensor described in the above embodiment can also comprise:immobilizing one or more specific binding substances in one or moredistinct locations defining an array within a well of a microtiterplate, wherein the distinct locations defining the array are locatedupon the surface of a colorimetric resonant reflectance opticalbiosensor which comprises an internal surface of the well; detecting acolorimetric resonant reflectance optical PWV for one or more distinctlocations within the well; applying one or more cleaving molecules tothe well; detecting a colorimetric resonant reflectance optical PWV forone or more distinct locations within the well; and comparing theinitial PWV above with the subsequent PWV above. The cleavage of one ormore entire specific binding substances at the one or more distinctlocations within the well is detected, and a peak wavelength value (PWV)is a relative measure of the specific binding substance that is bound tothe biosensor.

Another embodiment of the invention provides a method of detectinginhibition activity of one or more molecules against enzymes or bindingpartners that affect or bind specific binding substances, wherein thespecific binding substances are immobilized on a surface of acolorimetric resonant reflectance optical biosensor. The methodcomprises detecting a colorimetric resonant reflectance optical PWV of adistinct location; applying one or more molecules suspected of havinginhibition activity to the distinct location; applying one or moreenzymes or binding partners to the distinct location; detecting thecolorimetric resonant reflectance optical PWV of the distinct location;and comparing the initial PWV above with the subsequent PWV above.Alternatively, the one or more molecules suspected of having inhibitionactivity can be mixed with the one or more enzyme or binding partners,which, together, can be applied to the distinct location. The inhibitionactivity of one or more molecules against enzymes or binding partnerswhich effect or bind one or more specific binding substance is detected.A decrease in the initial colorimetric resonant reflectance optical PWVabove in relation to the subsequent colorimetric resonant reflectanceoptical PWV above is (1) a relative measure of the proportion ofspecific binding substance that is released from the biosensor surfaceor binding partners bound to the surface of the biosensor or (2) ameasure of relative effectiveness of one or more molecules suspected ofhaving inhibition activity.

The method of detecting inhibition activity of one or more moleculesagainst enzyme or binding partners which cleave specific bindingsubstances immobilized on a surface of a colorimetric resonantreflectance optical biosensor can also comprise: immobilizing one ormore specific binding substances in one or more distinct locationsdefining an array within a well of a microtiter plate, wherein thedistinct locations defining an array are located upon the surface of acolorimetric resonant reflectance optical biosensor which comprises aninternal surface of the well; detecting a colorimetric resonantreflectance optical PWV for the one or more distinct locations withinthe well; applying one or more molecules suspected of having inhibitionactivity to the well; applying one or more enzyme or binding partners tothe well; detecting a colorimetric resonant reflectance optical PWV forthe one or more distinct locations within the well; and comparing theinitial PWV above with the subsequent PWV above. Alternatively, the oneor more molecules suspected of having inhibition activity can be mixedwith the one or more enzymes or binding partners, which, together, canbe applied to the well. The inhibition activity of one or more moleculesagainst enzymes or binding partners which cleave one or more specificbinding substances at each distinct location within a well is detected.

A further embodiment of the invention provides a method of detecting achange in cell growth patterns. The method comprises growing cells on acolorimetric resonant reflectance optical biosensor; detecting acolorimetric resonant reflectance optical PWV; applying a test reagentto the cells; detecting the colorimetric resonant reflectance opticalPWV; and comparing the initial PWV above with the subsequent PWV above.The difference between the initial colorimetric resonant reflectanceoptical PWV above in relation to the subsequent colorimetric resonantreflectance optical PWV above indicates a change in cell growthpatterns.

In addition to the use of a single cell type in the embodiment thatprovides a method of detecting a change in cell growth pattern, two ormore different types of cells can be grown on the biosensor, wherein oneor more types of cells are grown in a well of the microtiter plate. Thechange in cell growth pattern can be selected from the group consistingof cell morphology, cell adhesion, cell migration, cell proliferationand cell death.

A still further embodiment of the invention provides a method ofdetecting molecules released from cells grown in a semi-permeableinternal sleeve held in contact with a colorimetric resonant reflectanceoptical biosensor. The method comprises detecting a colorimetricresonant reflectance optical PWV of the distinct location; growing cellsin the semi-permeable internal sleeve held in contact with thecolorimetric resonant reflectance optical biosensor at the distinctposition; detecting the colorimetric resonant reflectance optical PWV ofthe distinct location; and comparing the initial PWV above with thesubsequent PWV above. The binding of molecules released from cells grownin the semi-permeable internal sleeve held in contact with thecolorimetric resonant reflectance optical biosensor to the one or morespecific binding substances is detected. The initial peak wavelengthvalue (PWV) above is a relative measure of the specific bindingsubstance that is bound to the biosensor, and the difference between theinitial resonant optical biosensor PWV above in relation to thesubsequent resonant optical biosensor PWV above is a relative measure ofthe molecules released from cells grown in a semi-permeable internalsleeve that are bound to the specific binding substances.

The semi-permeable internal sleeve is a removable porous ornon-removable porous insert that is held in contact with the surface ofa biosensor, wherein the sleeve is permeable to molecules secreted fromthe cells cultured on its surface and wherein the sleeve is impermeableto whole cells.

The method of detecting molecules released from cells grown in asemi-permeable internal sleeve held in contact with a colorimetricresonant reflectance optical biosensor can also comprise: immobilizingone or more binding substances in one or more distinct locationsdefining an array within a well of a microtiter plate, wherein acolorimetric resonant reflectance optical biosensor comprises aninternal surface of the well; detecting a colorimetric resonantreflectance optical PWV for the one or more distinct locations definingan array within the well; growing cells in a semi-permeable internalsleeve held in contact with the well; detecting the colorimetricresonant reflectance optical PWV for the one or more distinct locationswithin the well; and comparing the initial PWV above with the subsequentPWV above. The difference between the initial colorimetric resonantreflectance optical PWV above in relation to the subsequent colorimetricresonant reflectance optical PWV above indicates the relative binding ofone or more molecules secreted from the cells growing on thesemi-permeable internal sleeve within a well to the one or more specificbinding substances immobilized at distinct locations within the well onthe surface of a colorimetric resonant reflectance optical biosensor.

Therefore, unlike methods for assays for surface plasmon resonance,resonant mirrors, and waveguide biosensors, the described methods enablemany thousands of individual binding reactions to take placesimultaneously upon the resonant optical biosensor surface. Suchhigh-throughput capabilities are highlighted particularly when thebiosensor surface comprises an interior surface of a microtiter platewell. In such an embodiment, thousands of assays can be performedsimultaneously in each of the wells of a standard microtiter plateformat, such as 2, 6, 8, 24, 48, 96, 384, 1536 or 3456 well formats.Clearly, this technology is useful in applications where large numbersof biomolecular interactions are measured in parallel, particularly whenmolecular labels will alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by this approach.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a cross-sectional view of a biosensor wherein light isshown as illuminating the bottom of the biosensor; however, light canilluminate the biosensor from either the top or the bottom. FIG. 1Bshows a diagram of a biosensor wherein light is shown as illuminatingthe bottom of the biosensor; however, light can illuminate the biosensorfrom either the top or the bottom;

FIG. 2 shows an embodiment of a colorimetric resonant reflectionbiosensor comprising a one-dimensional grating made according to themethods and compositions of the invention.

FIG. 3A-B shows a grating comprising a rectangular grid of squares (FIG.3A) or holes (FIG. 3B).

FIG. 4 shows a biosensor cross-section profile utilizing a sinusoidallyvarying grating profile.

FIG. 5 shows a resonant reflection or transmission filter structureconsisting of a set of concentric rings.

FIG. 6 shows a resonant reflective or transmission filter structurecomprising a hexagonal grid of holes (or a hexagonal grid of posts) thatclosely approximates the concentric circle structure of FIG. 5 withoutrequiring the illumination beam to be centered upon any particularlocation of the grid.

FIG. 7 shows a graphic representation of how adsorbed material, such asa protein monolayer, will increase the reflected wavelength of abiosensor that comprises a three-dimensional grating.

FIG. 8 shows three types of surface activation chemistry (Amine,Aldehyde, and Nickel) with corresponding chemical linker molecules thatcan be used to covalently attach various types of biomolecule receptorsto a biosensor.

FIG. 9A-C shows methods that can be used to amplify the mass of abinding partner such as detected DNA or detected protein on the surfaceof a biosensor.

FIG. 10 shows resonance wavelength of a biosensor as a function ofincident angle of detection beam.

FIG. 11 shows an example of the use of two coupled fibers to illuminateand collect reflected light from a biosensor.

FIG. 12 shows an example of the use of a beam splitter to enableilluminating and reflected light to share a common collimated opticalpath to a biosensor.

FIG. 13 shows a schematic diagram of a detection system.

FIG. 14 demonstrates an example of a biosensor that occurs on the tip ofa fiber probe for in vivo detection of biochemical substances.

FIG. 15 shows dependence of peak resonance wavelength on theconcentration of BSA dissolved in PBS, which was then allowed to dry ona biosensor surface.

FIG. 16A-B. FIG. 16A shows results of streptavidin detection at variousconcentrations for a biosensor that has been activated with NH₂ surfacechemistry linked to a biotin receptor molecule. FIG. 16B shows aschematic demonstration of molecules bound to a biosensor.

FIG. 17A-B. FIG. 17A shows an assay for detection of anti-goat IgG usinga goat antibody receptor molecule. BSA blocking of a detection surfaceyields a clearly measurable background signal due to the mass of BSAincorporated on the biosensor. A 66 nM concentration of anti-goat IgG iseasily measured above the background signal. FIG. 17B shows a schematicdemonstration of molecules bound to a biosensor.

FIG. 18A-B. FIG. 18A shows a nonlabeled ELISA assay for interferon-gamma(INF-gamma) using an anti-human IgG INF-gamma receptor molecule, and aneural growth factor (NGF) negative control. FIG. 18B shows a schematicdemonstration of molecules bound to a biosensor.

FIG. 19A-B. FIG. 19A shows detection of a 5-amino acid peptide (MW=860)and subsequent cleavage of a pNA label (MW=130) using enzyme caspase-3.FIG. 19B shows a schematic demonstration of molecules bound to abiosensor.

FIG. 20A-B. FIG. 20A shows resonant peak in liquid during continuousmonitoring of the binding of three separate protein layers. FIG. 20Bshows a schematic demonstration of molecules bound to a biosensor.

FIG. 21A-B. FIG. 21A shows endpoint resonant frequencies mathematicallydetermined from the data shown in FIG. 21. FIG. 21B shows a schematicdemonstration of molecules bound to a biosensor.

FIG. 22A-B. FIG. 22A shows kinetic binding measurement of IgG binding.FIG. 22B shows a schematic demonstration of molecules bound to abiosensor.

FIG. 23A-B. FIG. 23A shows kinetic measurement of a protease thatcleaves bound protein from a biosensor surface. FIG. 23B shows aschematic demonstration of molecules bound to a biosensor.

FIG. 24 shows a plot of the peak resonant wavelength values for testsolutions. The avidin solution was taken as the baseline reference forcomparison to the Avidin+BSA and Avidin+b−BSA solutions. Addition of BSAto avidin results in only a small resonant wavelength increase, as thetwo proteins are not expected to interact. However, because biotin andavidin bind strongly (Kd=10⁻¹⁵M), the avidin+b−BSA solution will containlarger bound protein complexes. The peak resonant wavelength value ofthe avidin+b−BSA solution thus provides a large shift compared toavidin+BSA.

FIG. 25 shows the PWV shift-referenced to a sensor with no chemicalfunctional groups immobilized, recorded due to the attachment of NH₂,NH₂+(NHS-PEG), and NH₂+(NHS-PEG-Biotin) molecules to the sensor surface.The error bars indicate the standard deviation of the recorded PWV shiftover 7 microtiter plate wells. The data indicates that the sensor candifferentiate between a clean surface, and one with immobilized NH₂, aswell as clearly detecting the addition of the NHS-PEG (molecular weightapproximately 2000 Daltons) molecule. The difference between surfaceimmobilized NHS-PEG and NHS-PEG-Biotin (molecular weight approximately3400 Dalton) is also measurable.

FIG. 26A-C shows the PWV shift response as a function of time forbiosensor wells when exposed to various concentrations of anti-biotinIgG (0–80 mg/ml) and allowed to incubate for 20 minutes. The NHS-PEGsurface (FIG. 26B) provides the lowest response, while theamine-activated surface (FIG. 26A) demonstrates a low level ofnonspecific interaction with the anti-biotin IgG at high concentrations.The NHS-PEG-Biotin surface (FIG. 26C) clearly demonstrates strongspecific interaction with the anti-biotin IgG—providing strong PWVshifts in proportion to the concentration of exposed anti-biotin IgG.

FIG. 27 shows the PWV shift magnitudes after 20 minutes from FIG. 26Cplotted as a function of anti-biotin IgG concentration in FIG. 26. Aroughly linear correlation between the IgG concentration and themeasured PWV shift is observed, and the lowest concentration IgGsolution (1.25 mg/ml, 8.33 nM) is clearly measurable over the negativecontrol PBS solution.

FIG. 28 shows the results of a cell morphology assay utilizingchondrocyte cells grown on the colorimetric resonant reflectance opticalbiosensor. The cells were observed to remain attached to the surface ofthe biosensor throughout the assay; the observed decrease in PWV uponaddition of 2 mg/ml trypsin to the biosensor wells containing thechondrocyte cells indicates a change in chondrocyte cell morphology.

FIG. 29 shows the results of a cell adhesion assay using kidney tumorcells. Trypsin was added to six biosensor wells containing kidney tumorcells grown on the surface of the biosensor. Two wells were utilized asreplicate samples for each of the three trypsin concentrations. Uponaddition of the trypsin, a decrease in PWV is observed indicating thedetachment of the cells from the surface of the sensor.

FIG. 30 shows an example of a system for angular scanning of abiosensor.

FIG. 31 shows an example of a biosensor used as a microarray.

FIG. 32A-B shows two biosensor formats that can incorporate acolorimetric resonant reflectance biosensor. FIG. 32A shows a biosensorthat is incorporated into a microtiter plate. FIG. 32B shows a biosensorin a microarray slide format.

FIG. 33 shows an array of arrays concept for using a biosensor platformto perform assays with higher density and throughput.

FIG. 34A-B. FIG. 34A shows a measure resonant wavelength shift caused byattachment of a strepavidin receptor layer and subsequent detection of abiotinylated IgG. FIG. 34B shows a schematic demonstration of moleculesbound to biosensor.

FIG. 35A-B. FIG. 35A shows the spotting of rabbit, chicken, goat, andhuman IgG on a colorimetric resonant reflectance biosensor microarray.FIG. 35B shows the result of flowing anti-human-IgG over the sensorsurface, indicating greater binding between the human-IgG and theanti-human-IgG.

FIG. 36A-B. With Poly-T immobilized on the sensor surface, FIG. 36Ashows the different degrees of hybridization affinities between theimmobilized oligonucleotides with Poly-T, Poly-A, and T7-promoter. FIG.36-B shows the endpoint data with error bars.

FIG. 37 shows an example of the specificity with which protein-DNAinteractions, in this case between T7-promoter DNA and T7 RNApolymerase, can be detected.

DETAILED DESCRIPTION OF THE INVENTION

A colorimetric resonant reflectance optical biosensor allows biochemicalinteractions to be measured on the biosensor's surface without the useof fluorescent tags, colorimetric labels or any other type of tag orlabel. A biosensor surface contains an optical structure that, whenilluminated with collimated white light, is designed to reflect only anarrow band of wavelengths. The narrow wavelength band is described as awavelength “peak.” The “peak wavelength value” (PWV) changes whenmaterials, such as biological materials, are deposited or removed fromthe biosensor surface. A readout instrument is used to illuminatedistinct locations on a biosensor surface with collimated white light,and to collect collimated reflected light. The collected light isgathered into a wavelength spectrometer for determination of PWV.

A biosensor structure can be incorporated into standard disposablelaboratory items such as microtiter plates by bonding the structure(biosensor side up) into the bottom of a bottomless microtiter platecartridge. Incorporation of a biosensor into common laboratory formatcartridges is desirable for compatibility with existing microtiter platehandling equipment such as mixers, incubators, and liquid dispensingequipment.

The functional advantages of each of the assay methods defined in thisdisclosure arise from the properties of a colorimetric resonantreflectance biosensor. First, biochemical interactions are measuredwithout the use of labels. Second, many interactions can be monitoredsimultaneously. Third, the biosensor is incorporated into a standardmicrotiter plate for isolation and liquid containment of parallelassays.

For the majority of assays currently performed for genomics, proteomics,pharmaceutical compound screening, and clinical diagnostic applications,fluorescent or colorimetric chemical labels are commonly attached to themolecules under study so they may be readily visualized. Becauseattachment of a label substantially increases assay complexity andpossibly alters the functionality of molecules through conformationalmodification or epitope blocking, various label-free biosensortechnologies have emerged. Label-free detection phenomenologies includemeasuring changes in mass, microwave transmission line characteristics,microcantilever deflection, or optical density upon a surface that isactivated with a receptor molecule with high affinity for a detectedmolecule. The widespread commercial acceptance of label-free biosensortechnologies has been limited by their ability to provide high detectionsensitivity and high detection parallelism in a format that isinexpensive to manufacture and package. For example, biosensorsfabricated upon semiconductor or glass wafers in batch photolithography,etch and deposition processes are costly to produce and package if thebiosensor area is to be large enough to contain large numbers ofparallel assays. Similarly, the requirement of making electricalconnections to individual biosensors in an array poses difficultchallenges in terms of package cost and compatibility with exposure ofthe biosensor to fluids.

Definitions

Colorimetric Resonant Reflectance Optical Biosensor:

“Colorimetric resonant reflectance optical biosensors,” alternativelyreferred to herein as biosensors, are defined herein as subwavelengthstructured surface (SWS) biosensors and surface-relief volumediffractive (SRVD) biosensors. See, e.g., U.S. application Ser. No.10/059,060 entitled “Resonant Reflection Microarray.”

Entire Specific Binding Substance:

“Entire specific binding substance,” as used herein, refers tosubstantially the entirety of a molecule of interest, such that, forexample, the cleavage of substantially an entire specific bindingsubstance from the surface of a colorimetric resonant reflectanceoptical biosensor yields the substantially complete, native specificbinding substance.

Microtiter Plate:

“Microtiter plate,” as used herein, is defined as a microtiter ormultiwell plate of 2, 6, 8, 24, 48, 96, 384, 1536 or 3456 well formats,or any other number of wells.

Test Reagent:

“Test reagent,” as used herein, is defined as any enzyme or chemicalcompound and solutions thereof. Non-limiting examples of enzymes areproteases, lipases, nucleases, lyases, peptidases, hydrolases, ligases,kinases and phosphatases. In addition to the enzymes, chemical compoundsand solutions thereof, “test reagent” also refers to buffer blanksthereof. A buffer blank refers to reagents or solutions identical incomposition to those added to the other recited test reagents, with theenzyme component omitted.

Semi-Permeable Internal Sleeve:

A “semi-permeable internal sleeve,” alternatively referred to as“insert” or “sleeve” herein, is defined as a porous material that iscapable of supporting cell growth. A semi-permeable internal sleeve ispermeable to proteins or other molecules secreted, shed or otherwiseejected from the cell grown on the sleeve surface but impermeable to awhole cell. A semi-permeable internal sleeve is generally held a shortdistance from the surface of a biosensor to which specific bindingsubstances are bound or the growth media or buffer on the surface of abiosensor such that free diffusion of the secreted, shed or otherwiseejected moieties can occur through the sleeve. A semi-permeable internalsleeve can reside on any kind of colorimetric resonant reflectanceoptical biosensor, as defined above, within or without a well of amicrotiter plate.

A semi-permeable internal sleeve that is “held in contact” with asurface of a biosensor or the surface of growth media or buffer on thesurface of a biosensor is defined herein as (1) being positioned suchthat the sleeve is in close proximity to, but not in direct physicalcontact with the surface of the biosensor; (2) being positioned suchthat the sleeve is in physical contact with the surface of the buffer orgrowth media that is positioned on the biosensor surface; or (3) beingpositioned or connected in any manner such that diffusion of moleculessecreted, shed or otherwise ejected from the cells through thesemi-permeable internal sleeve is facilitated and, preferably,unhindered. “Held in contact” is also referred to as sitting or fitting“adjacent to the biosensor surface.”

The types of material used as semi-permeable internal sleeve can be, forexample, polyethylene terephthalate (PET) or polytetrafluoroethylene(PTFE) such as the material used within commercially available cellculture inserts (BD Falcon, Millipore).

Inhibition Activity:

“Inhibition activity” is defined herein as the ability of a molecule orcompound to slow or stop another molecule from carrying out catalyticactivity. For example, a compound that has inhibition activity of aprotease inhibits the protease from cleaving a protein. Such inhibitionactivity is carried out “against” the catalytic molecule. “Inhibitionactivity” also means the ability of a molecule or compound tosubstantially inhibit or partially inhibit the binding of a bindingpartner to a specific binding substance.

Nucleic Acid:

“Nucleic acid” is defined herein as single or double stranded polymersof natural or non-natural nucleotides or derivatives thereof, linked by3′,5′ phosphodiester linkages.

Oligonucleotide:

“Oligonucleotide” is defined herein as a single or double strandedpolymer sequence of natural or non-natural nucleotides or derivativesthereof joined by phosphodiester bonds. “Oligonucleotide” generallyrefers to short polynucleotides of a length approximately 20 bases orless, beyond which they are preferentially referred to apolynucleotides.

Protein:

“Protein” is defined herein as a linear polymer of natural ornon-natural amino acids or derivatives thereof joined by peptide bondsin a specific sequence.

Peptide:

“Peptide” is defined herein as any of a class of molecules thathydrolyze into amino acids and form the basic building blocks ofproteins. Generally refers to a short polypeptide or protein fragment.

Combinatorial Chemical Library:

“Combinatorial chemical library” is defined herein as a diverse set ofmolecules resulting from the combination of their constituent buildingblock materials in myriad ways.

Cell Membrane:

“Cell membrane” is defined herein as the external, limiting lipidbilayer membrane of cells.

Tissue:

“Tissue” is defined herein as a group of cells, often of mixed types andusually held together by extracellular matrix, that perform a particularfunction. Also, in a more general sense, “tissue” can refer to thebiological grouping of a cell type result from a common factor; forexample, connective tissue, where the common feature is the function orepithelial tissue, where the common factor is the pattern oforganization.

Receptor:

“Receptor” is defined herein as a membrane-bound or membrane-enclosedmolecule that binds to, or responds to something more mobile (theligand), with high specificity.

Ligand:

“Ligand” is defined herein as a molecule that binds to another; innormal usage a soluble molecule, such as a hormone or neurotransmitter,that binds to a receptor. Also analogous to “binding substance” herein.

Cytokine:

“Cytokine” is defined herein as proteins released by cells and thataffect the behavior of other cells. Similar to “hormone”, but the termtends to be used as a generic word for interleukins, lymphokines andseveral related signaling molecules such as TNF and interferons.

Chemokine:

“Chemokine” is defined herein as small secreted proteins that stimulatechemotaxis of leucocytes.

Extracellular Matrix Material:

“Extracellular Matrix Material” is defined herein as any materialproduced by cells and secreted into the surrounding medium, but usuallyapplied to the non-cellular portion of animal tissues.

Antigen:

“Antigen” is defined herein as a substance inducing an immune response.The antigenic determinant group is termed an epitope, and the epitope inthe context of a carrier molecule (that can optionally be part of thesame molecule, for example, botulism neurotoxin A, a single molecule,has three different epitopes. See Mullaney et al., Infect Immun October2001; 69(10): 6511–4) makes the carrier molecule active as an antigen.Usually antigens are foreign to the animal in which they produce immunereactions.

Polyclonal Antibody:

“Polyclonal antibody” is defined herein as an antibody produced byseveral clones of B-lymphocytes as would be the case in a whole animal.Usually refers to antibodies raised in immunized animals.

Monoclonal Antibody:

“Monoclonal antibody” is defined herein as a cell line, whether withinthe body or in culture, that has a single clonal origin. Monoclonalantibodies are produced by a single clone of hybridoma cells, and aretherefore a single species of antibody molecule.

Single Chain Antibody (Scfv):

“Single chain antibody (Scfv)” is defined herein as a recombinant fusionprotein wherein the two antigen binding regions of the light and heavychains (Vh and Vl) are connected by a linking peptide, which enables theequal expression of both the light and heavy chains in a heterologousorganism and stabilizes the protein.

F(Ab) Fragment:

“F(Ab) fragment” is defined herein as fragments of immunoglobulinprepared by papain treatment. Fab fragments consist of one light chainlinked through a disulphide bond to a portion of the heavy chain, andcontain one antigen binding site. They can be considered as univalentantibodies.

F(Ab′)2 Fragments:

“F(Ab′)2 Fragments” is defined herein as the approximately 90 kDaprotein fragment obtained upon pepsin hydrolysis of an immunoglobulinmolecule N-terminal to the site of the pepsin attack. Contains both Fabfragments held together by disulfide bonds in a short section of the Fefragment.

Fv Fragments:

“Fv Fragments” is defined herein as the N-terminal portion of a Fabfragment of an immunoglobulin molecule, consisting of the variableportions of one light chain and one heavy chain.

Small Organic Molecules:

“Small Organic Molecules” is defined herein as any smallcarbon-containing molecule that is not otherwise classified as one ofthe above-defined organic molecules, such as, for example, apolypeptide.

Subwavelength Structured Surface (SWS) Biosensor

In one embodiment of the invention, a subwavelength structured surface(SWS) is used to create a sharp optical resonant reflection at aparticular wavelength that can be used to track with high sensitivitythe interaction of biological materials, such as specific bindingsubstances or binding partners or both. A colorimetric resonantreflectance diffractive grating surface acts as a surface bindingplatform for specific binding substances.

Subwavelength structured surfaces are an unconventional type ofdiffractive optic that can mimic the effect of thin-film coatings. (Peng& Morris, “Resonant scattering from two-dimensional gratings,” J. Opt.Soc. Am. A, Vol. 13, No. 5, p. 993, May 1996; Magnusson, & Wang, “Newprinciple for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022,August, 1992; Peng & Morris, “Experimental demonstration of resonantanomalies in diffraction from two-dimensional gratings,” Optics Letters,Vol. 21, No. 8, p. 549, April, 1996). A SWS structure contains aone-dimensional, two-dimensional, or three dimensional grating in whichthe grating period is small compared to the wavelength of incident lightso that no diffractive orders other than the reflected and transmittedzeroth orders are allowed to propagate. A SWS surface narrowband filtercan comprise a grating sandwiched between a substrate layer and a coverlayer that fills the grating grooves. Optionally, a cover layer is notused. When the effective index of refraction of the grating region isgreater than the substrate or the cover layer, a waveguide is created.When a filter is designed properly, incident light passes into thewaveguide region and propagates as a leaky mode. A grating structureselectively couples light at a narrow band of wavelengths into thewaveguide. The light propagates only a very short distance (on the orderof 10–100 micrometers), undergoes scattering, and couples with theforward- and backward-propagating zeroth-order light. This highlysensitive coupling condition can produce a resonant grating effect onthe reflected radiation spectrum, resulting in a narrow band ofreflected or transmitted wavelengths. The depth and period of thegrating are less than the wavelength of the resonant grating effect.

The reflected or transmitted color of this structure can be modulated bythe addition of molecules such as specific binding substances or bindingpartners or both to the upper surface of the cover layer or the gratingsurface. The added molecules increase the optical path length ofincident radiation through the structure, and thus modify the wavelengthat which maximum reflectance or transmittance will occur.

In one embodiment, a biosensor, when illuminated with white light, isdesigned to reflect only a single wavelength or a narrow band ofwavelengths. When specific binding substances are attached to thesurface of the biosensor, the reflected wavelength is shifted due to thechange of the optical path of light that is coupled into the grating. Bylinking specific binding substances to a biosensor surface,complementary binding partner molecules can be detected without the useof any kind of fluorescent probe, particle label or any other type oflabel. The detection technique is capable of resolving changes of, forexample, ˜0.1 nm thickness of protein binding, and can be performed withthe biosensor surface either immersed in fluid or dried.

A detection system consists of, for example, a light source thatilluminates a small spot of a biosensor at normal incidence through, forexample, a fiber optic probe, and a spectrometer that collects thereflected light through, for example, a second fiber optic probe also atnormal incidence. Because no physical contact occurs between theexcitation/detection system and the biosensor surface, no specialcoupling prisms are required and the biosensor can be easily adapted toany commonly used assay platform including, for example, microtiterplates and microarray slides. A single spectrometer reading can beperformed in several milliseconds, thus it is possible to quicklymeasure a large number of molecular interactions taking place inparallel upon a biosensor surface, and to monitor reaction kinetics inreal time.

This technology is useful in applications where large numbers ofbiomolecular interactions are measured in parallel, particularly whenmolecular labels would alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by the compositionsand methods of the invention.

FIGS. 1A and 1B are diagrams of an example of a colorimetric resonantreflection diffractive grating biosensor. In FIG. 1, n_(substrate)represents a substrate material. n₂ represents the refractive index ofan optical grating. n₁ represents an optional cover layer. n_(bio)represents the refractive index of one or more specific bindingsubstances. t₁ represents the thickness of the optional cover layerabove the one-, two- or three-dimensional grating structure. t₂represents the thickness of the grating. t_(bio) represents thethickness of the layer of one or more specific binding substances. Inone embodiment, are n2<n1 (see FIG. 1A). Layer thicknesses (i.e. coverlayer, one or more specific binding substances, or an optical grating)are selected to achieve resonant wavelength sensitivity to additionalmolecules on the top surface. The grating period is selected to achieveresonance at a desired wavelength.

A SWS biosensor comprises an optical grating comprised of a highrefractive index material, a substrate layer that supports the grating,and one or more specific binding substances immobilized on the surfaceof the grating opposite of the substrate layer. Optionally, a coverlayer covers the grating surface. An optical grating made according tothe invention is coated with a high refractive index dielectric filmwhich can be comprised of a material that includes, for example, zincsulfide, titanium dioxide, tantalum oxide, and silicon nitride. Across-sectional profile of a grating with optical features can compriseany periodically repeating function, for example, a “square-wave.” Anoptical grating can also comprise a repeating pattern of shapes selectedfrom the group consisting of lines, squares, circles, ellipses,triangles, trapezoids, sinusoidal waves, ovals, rectangles, andhexagons. A biosensor of the invention can also comprise an opticalgrating comprised of, for example, plastic or epoxy, which is coatedwith a high refractive index material.

Sensor Characteristics

Linear gratings (i.e., one dimensional gratings) have resonantcharacteristics where the illuminating light polarization is orientedperpendicular to the grating period. A schematic diagram of oneembodiment a linear grating structure with an optional cover layer isshown in FIG. 2. A colorimetric resonant reflection biosensor can alsocomprise, for example, a two-dimensional grating, e.g., a hexagonalarray of holes (see FIG. 3B) or squares (see FIG. 3A). Other shapes canbe used as well. A linear grating has the same pitch (i.e. distancebetween regions of high and low refractive index), period, layerthicknesses, and material properties as a hexagonal array grating.However, light must be polarized perpendicular to the grating lines inorder to be resonantly coupled into the optical structure. Therefore, apolarizing filter oriented with its polarization axis perpendicular tothe linear grating must be inserted between the illumination source andthe biosensor surface. Because only a small portion of the illuminatinglight source is correctly polarized, a longer integration time isrequired to collect an equivalent amount of resonantly reflected lightcompared to a hexagonal grating.

An optical grating can also comprise, for example, a “stepped” profile,in which high refractive index regions of a single, fixed height areembedded within a lower refractive index cover layer. The alternatingregions of high and low refractive index provide an optical waveguideparallel to the top surface of the biosensor.

It is also possible to make a resonant biosensor in which the highrefractive index material is not stepped, but which varies with lateralposition. FIG. 4 shows a profile in which the high refractive indexmaterial of the two-dimensional grating, n₂, is sinusoidally varying inheight. To produce a resonant reflection at a particular wavelength, theperiod of the sinusoid is identical to the period of an equivalentstepped structure. The resonant operation of the sinusoidally varyingstructure and its functionality as a biosensor has been verified usingGSOLVER (Grating Solver Development Company, Allen, Tex., USA) computermodels.

A biosensor of the invention can further comprise a cover layer on thesurface of an optical grating opposite of a substrate layer. Where acover layer is present, the one or more specific binding substances areimmobilized on the surface of the cover layer opposite of the grating.Preferably, a cover layer comprises a material that has a lowerrefractive index than a material that comprises the grating. A coverlayer can be comprised of, for example, glass (including spin-on glass(SOG)), epoxy, or plastic.

For example, various polymers that meet the refractive index requirementof a biosensor can be used for a cover layer. SOG can be used due to itsfavorable refractive index, ease of handling, and readiness of beingactivated with specific binding substances using the wealth of glasssurface activation techniques. When the flatness of the biosensorsurface is not an issue for a particular system setup, a gratingstructure of SiN/glass can directly be used as the sensing surface, theactivation of which can be done using the same means as on a glasssurface.

Resonant reflection can also be obtained without a planarizing coverlayer over an optical grating. For example, a biosensor can contain onlya substrate coated with a structured thin film layer of high refractiveindex material. Without the use of a planarizing cover layer, thesurrounding medium (such as air or water) fills the grating. Therefore,specific binding substances are immobilized to the biosensor on allsurfaces of an optical grating exposed to the specific bindingsubstances, rather than only on an upper surface.

In general, a biosensor of the invention will be illuminated with whitelight that will contain light of every polarization angle. Theorientation of the polarization angle with respect to repeating featuresin a biosensor grating will determine the resonance wavelength. Forexample, a “linear grating” (i.e., a one-dimensional grating) biosensorconsisting of a set of repeating lines and spaces will have two opticalpolarizations that can generate separate resonant reflections. Lightthat is polarized perpendicularly to the lines is called “s-polarized,”while light that is polarized parallel to the lines is called“p-polarized.” Both the s and p components of incident light existsimultaneously in an unfiltered illumination beam, and each generates aseparate resonant signal. A biosensor can generally be designed tooptimize the properties of only one polarization (the s-polarization),and the non-optimized polarization is easily removed by a polarizingfilter.

In order to remove the polarization dependence, so that everypolarization angle generates the same resonant reflection spectra, analternate biosensor structure can be used that consists of a set ofconcentric rings. In this structure, the difference between the insidediameter and the outside diameter of each concentric ring is equal toabout one-half of a grating period. Each successive ring has an insidediameter that is about one grating period greater than the insidediameter of the previous ring. The concentric ring pattern extends tocover a single sensor location—such as an array spot or a microtiterplate well. Each separate microarray spot or microtiter plate well has aseparate concentric ring pattern centered within it. See, e.g., FIG. 5.All polarization directions of such a structure have the samecross-sectional profile. The concentric ring structure must beilluminated precisely on-center to preserve polarization independence.The grating period of a concentric ring structure is less than thewavelength of the resonantly reflected light. The grating period isabout 0.01 micron to about 1 micron. The grating depth is about 0.01 toabout 1 micron.

In another embodiment, an array of holes or posts are arranged toclosely approximate the concentric circle structure described abovewithout requiring the illumination beam to be centered upon anyparticular location of the grid. See e.g. FIG. 6. Such an array patternis automatically generated by the optical interference of three laserbeams incident on a surface from three directions at equal angles. Inthis pattern, the holes (or posts) are centered upon the corners of anarray of closely packed hexagons as shown in FIG. 6. The holes or postsalso occur in the center of each hexagon. Such a hexagonal grid of holesor posts has three polarization directions that “see” the samecross-sectional profile. The hexagonal grid structure, therefore,provides equivalent resonant reflection spectra using light of anypolarization angle. Thus, no polarizing filter is required to removeunwanted reflected signal components. The period of the holes or postscan be about 0.01 microns to about 1 micron and the depth or height canbe about 0.01 microns to about 1 micron.

Another grating that can be produced using the methods of the inventionis a volume surface-relief volume diffractive grating (a SRVD grating),also referred to as a three-dimensional grating. SRVD gratings have asurface that reflects predominantly at a particular narrow band ofoptical wavelengths when illuminated with a broad band of opticalwavelengths. Where specific binding substances and/or binding partnersare immobilized on a SRVD grating, producing a SRVD biosensor, thereflected narrow band of wavelengths of light is shifted.One-dimensional surfaces, such as thin film interference filters andBragg reflectors, can select a narrow range of reflected or transmittedwavelengths from a broadband excitation source, however, the depositionof additional material, such as specific binding substances and/orbinding partners onto their upper surface results only in a change inthe resonance linewidth, rather than the resonance wavelength. Incontrast, SRVD biosensors have the ability to alter the reflectedwavelength with the addition of material, such as specific bindingsubstances and/or binding partners to the surface. The depth and periodof relief volume diffraction structures are less than the resonancewavelength of light reflected from a biosensor.

A three-dimensional surface-relief volume diffractive grating can be,for example, a three-dimensional phase-quantized terraced surface reliefpattern whose groove pattern resembles a stepped pyramid. When such agrating is illuminated by a beam of broadband radiation, light will becoherently reflected from the equally spaced terraces at a wavelengthgiven by twice the step spacing times the index of refraction of thesurrounding medium. Light of a given wavelength is resonantly diffractedor reflected from the steps that are a half-wavelength apart, and with abandwidth that is inversely proportional to the number of steps.

An example of a three-dimensional phase-quantized terraced surfacerelief pattern is a pattern that resembles a stepped pyramid. Eachinverted pyramid is approximately 1 micron in diameter, preferably, eachinverted pyramid can be about 0.5 to about 5 microns diameter, includingfor example, about 1 micron. The pyramid structures can be close-packedso that a typical microarray spot with a diameter of about 150–200microns can incorporate several hundred stepped pyramid structures. Therelief volume diffraction structures have a period of about 0.1 to about1 micron and a depth of about 0.1 to about 1 micron. FIG. 7 demonstrateshow individual microarray locations (with an entire microarray spotincorporating hundreds of pyramids now represented by a single pyramidfor one microarray spot) can be optically queried to determine ifspecific binding substances or binding partners are adsorbed onto thesurface. When the biosensor is illuminated with white light, pyramidstructures without significant bound material will reflect wavelengthsdetermined by the step height of the pyramid structure. When higherrefractive index material, such as binding partners or specific bindingsubstances, are incorporated over the reflective metal surface, thereflected wavelength is modified to shift toward longer wavelengths. Thecolor that is reflected from the terraced step structure istheoretically given as twice the step height times the index ofrefraction of a reflective material that is coated onto the firstsurface of a sheet material of a SRVD biosensor. A reflective materialcan be, for example silver, aluminum, or gold.

One or more specific binding substances, as described above, areimmobilized on the reflective material of a SRVD biosensor. One or morespecific binding substances can be arranged in an array of one or moredistinct locations, as described above, on the reflective material.

Because the reflected wavelength of light from a SRVD biosensor isconfined to a narrow bandwidth, very small changes in the opticalcharacteristics of the surface manifest themselves in easily observedchanges in reflected wavelength spectra. The narrow reflection bandwidthprovides a surface adsorption sensitivity advantage compared toreflectance spectrometry on a flat surface.

A SRVD biosensor reflects light predominantly at a first single opticalwavelength when illuminated with a broad band of optical wavelengths,and reflects light at a second single optical wavelength when one ormore specific binding substances are immobilized on the reflectivesurface. The reflection at the second optical wavelength results fromoptical interference. A SRVD biosensor also reflects light at a thirdsingle optical wavelength when the one or more specific bindingsubstances are bound to their respective binding partners, due tooptical interference.

Readout of the reflected color can be performed serially by focusing amicroscope objective onto individual microarray spots and reading thereflected spectrum, or in parallel by, for example, projecting thereflected image of the microarray onto a high resolution color CCDcamera.

In one embodiment of the invention, an optical device is provided. Anoptical device comprises a structure similar to a biosensor of theinvention; however, an optical device does not comprise one of morebinding substances immobilized on the grating. An optical device can beused as, for example, a narrow band optical filter.

Specific Binding Substances and Binding Partners

One or more specific binding substances are immobilized on the one- ortwo- or three-dimensional grating or cover layer, if present, by forexample, physical adsorption or by chemical binding. A specific bindingsubstance can be, for example, a nucleic acid, peptide, proteinsolutions, peptide solutions, single or double stranded DNA solutions,RNA solutions, RNA-DNA hybrid solutions, solutions containing compoundsfrom a combinatorial chemical library, antigen, polyclonal antibody,monoclonal antibody, single chain antibody (scFv), F(ab) fragment,F(ab′)₂ fragment, Fv fragment, small organic molecule, cell, virus,bacteria, polymer or biological sample. A biological sample can be forexample, blood, plasma, serum, gastrointestinal secretions, homogenatesof tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid,amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavagefluid, semen, lymphatic fluid, tears, or prostatic fluid. The polymer isselected from the group of long chain molecules with multiple activesites per molecule consisting of hydrogel, dextran, poly-amino acids andderivatives thereof, including poly-lysine (comprising poly-1-lysine andpoly-d-lysine), poly-phe-lysine and poly-glu-lysine.

Preferably, one or more specific binding substances are arranged in anarray of one or more distinct locations on a biosensor. An array ofspecific binding substances comprises one or more specific bindingsubstances on a surface of a biosensor of the invention such that asurface contains many distinct locations, each with a different specificbinding substance or with a different amount of a specific bindingsubstance. For example, an array can comprise 1, 10, 100, 1,000, 10,000or 100,000 distinct locations. Such a biosensor surface is called anarray because one or more specific binding substances are typically laidout in a regular grid pattern in x-y coordinates. However, an array ofthe invention can comprise one or more specific binding substance laidout in any type of regular or irregular pattern. For example, distinctlocations can define an array of spots of one or more specific bindingsubstances. An array spot can be about 50 to about 500 microns indiameter. An array spot can also be about 150 to about 200 microns indiameter. One or more specific binding substances can be bound to theirspecific binding partners.

An array on a biosensor of the invention can be created by placingmicrodroplets of one or more specific binding substances onto, forexample, an x-y grid of locations on a grating or cover layer surface.When the biosensor is exposed to a test sample comprising one or morebinding partners, the binding partners will be preferentially attractedto distinct locations on the microarray that comprise specific bindingsubstances that have high affinity for the binding partners. Some of thedistinct locations will gather binding partners onto their surface,while other locations will not.

A specific binding substance specifically binds to a binding partnerthat is added to the surface of a biosensor of the invention. A specificbinding substance specifically binds to its binding partner, but doesnot substantially bind other binding partners added to the surface of abiosensor. For example, where the specific binding substance is anantibody and its binding partner is a particular antigen, the antibodyspecifically binds to the particular antigen, but does not substantiallybind other antigens. A binding partner can be, for example, a nucleicacid, peptide, protein solutions, peptide solutions, single or doublestranded DNA solutions, RNA solutions, RNA-DNA hybrid solutions,solutions containing compounds from a combinatorial chemical library,antigen, polyclonal antibody, monoclonal antibody, single chain antibody(scFv), F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organicmolecule, cell, virus, bacteria, polymer or biological sample. Abiological sample can be, for example, blood, plasma, serum,gastrointestinal secretions, homogenates of tissues or tumors, synovialfluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinalfluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid,tears, and prostatic fluid.

One example of an array of the invention is a nucleic acid array, inwhich each distinct location within the array contains a differentnucleic acid molecule. In this embodiment, the spots within the nucleicacid microarray detect complementary chemical binding with an opposingstrand of a nucleic acid in a test sample.

While microtiter plates are the most common format used for biochemicalassays, microarrays are increasingly seen as a means for maximizing thenumber of biochemical interactions that can be measured at one timewhile minimizing the volume of precious reagents. By application ofspecific binding substances with a microarray spotter onto a biosensorof the invention, specific binding substance densities of 10,000specific binding substances/in² can be obtained. By focusing anillumination beam to interrogate a single microarray location, abiosensor can be used as a label-free microarray readout system.

Further, both the microarray and microtiter plate embodiments can becombined such that one or more specific binding substances are arrangedin an array of one or more distinct locations on the sensor surface,said surface residing within one or more wells of the microtiter plateand comprising one or more surfaces of the microtiter plate, preferablythe bottom surface. The array of specific binding substances comprisesone or more specific binding substances on the sensor surface within amicrotiter plate well such that a surface contains one or more distinctlocations, each with a different specific binding substance or with adifferent amount of a specific binding substance. For example, an arraycan comprise 1, 10, 100, 1,000, 10,000 or 100,000 distinct locations.Thus, each well of the microtiter plate embodiment can have within it anarray of one or more distinct locations separate from the other wells ofthe microtiter plate embodiment, which allows multiple different samplesto be processed on one microtiter plate of the invention, one or moresamples for each separate well. The array or arrays within any one wellcan be the same or different than the array or arrays found in any othermicrotiter wells of the same microtiter plate.

Immobilization or One or More Specific Binding Substances

Immobilization of one or more binding substances onto a biosensor isperformed so that a specific binding substance will not be washed awayby rinsing procedures, and so that its binding to binding partners in atest sample is unimpeded by the biosensor surface. Several differenttypes of surface chemistry strategies have been implemented for covalentattachment of specific binding substances to, for example, glass for usein various types of microarrays and biosensors. These same methods canbe readily adapted to a biosensor of the invention. Surface preparationof a biosensor so that it contains the correct functional groups forbinding one or more specific binding substances is an integral part ofthe biosensor manufacturing process.

One or more specific binding substances can be attached to a biosensorsurface by physical adsorption (i.e., without the use of chemicallinkers) or by chemical binding (i.e., with the use of chemical linkers)as well as electrochemical binding, electrostatic binding, hydrophobicbinding and hydrophilic binding. Chemical binding can generate strongerattachment of specific binding substances on a biosensor surface andprovide defined orientation and conformation of the surface-boundmolecules.

Several examples of chemical binding of specific binding substances to abiosensor of the invention appear in Example 2, below. Other types ofchemical binding include, for example, binding via the followingfunctional groups: an amine group, aldehyde group, nickel group, acidgroup, alkane group, alkene group, alkyne group, aromatic group, alcoholgroup, ether group, ketone group, ester group, amide group, amino acidgroup, nitro group, nitrile group, carbohydrate group, thiol group,organic phosphate group, lipid group, phospholipid group or steroidgroup. These surfaces can be used to attach several different types ofchemical linkers to a biosensor surface, as shown in FIG. 8. Forexample, an amine surface can be used to attach several types of linkermolecules while an aldehyde surface can be used to bind proteinsdirectly, without an additional linker. A nickel surface can be used tobind molecules that have an incorporated histidine (“his”) tag.Detection of “his-tagged” molecules with a nickel-activated surface iswell known in the art (Whitesides, Anal. Chem. 68, 490, (1996)).

Immobilization of specific binding substances to plastic, epoxy, or highrefractive index material can be performed essentially as described forimmobilization to glass. However, the acid wash step can be eliminatedwhere such a treatment would damage the material to which the specificbinding substances are immobilized.

For the detection of binding partners at concentrations less than about˜0.1 ng/ml, it is preferable to amplify and transduce binding partnersbound to a biosensor into an additional layer on the biosensor surface.The increased mass deposited on the biosensor can be easily detected asa consequence of increased optical path length. By incorporating greatermass onto a biosensor surface, the optical density of binding partnerson the surface is also increased, thus rendering a greater resonantwavelength shift than would occur without the added mass. The additionof mass can be accomplished, for example, enzymatically, through a“sandwich” assay, or by direct application of mass to the biosensorsurface in the form of appropriately conjugated beads or polymers ofvarious size and composition. This principle has been exploited forother types of optical biosensors to demonstrate sensitivity increasesover 1500× beyond sensitivity limits achieved without massamplification. See, e.g., Jenison et al., “Interference-based detectionof nucleic acid targets on optically coated silicon,” NatureBiotechnology, 19: 62–65, 2001.

As an example, FIG. 9A shows that an NH₂—activated biosensor surface canhave a specific binding substance comprising a single-strand DNA captureprobe immobilized on the surface. The capture probe interactsselectively with its complementary target binding partner. The bindingpartner, in turn, can be designed to include a sequence or tag that willbind a “detector” molecule. As shown in FIG. 9A, a detector molecule cancontain, for example, a linker to horseradish peroxidase (HRP) that,when exposed to the correct enzyme, will selectively deposit additionalmaterial on the biosensor only where the detector molecule is present.Such a procedure can add, for example, 300 angstroms of detectablebiomaterial to the biosensor within a few minutes.

A “sandwich” approach can also be used to enhance detection sensitivity.In this approach, a large molecular weight molecule can be used toamplify the presence of a low molecular weight molecule. For example, abinding partner with a molecular weight of, for example, about 0.1 kDato about 20 kDa, can be tagged with, for example,succinimidyl-6-[a-methyl-a-(2-pyridyl-dithio) toluamido] hexanoate(SMPT), or dimethylpimelimidate (DMP), histidine, or a biotin molecule,as shown in FIG. 9B. Where the tag is biotin, the biotin molecule willbinds strongly with streptavidin, which has a molecular weight of 60kDa. Because the biotin/streptavidin interaction is highly specific, thestreptavidin amplifies the signal that would be produced only by thesmall binding partner by a factor of 60.

Detection sensitivity can be further enhanced through the use ofchemically derivatized small particles. “Nanoparticles” made ofcolloidal gold, various plastics, or glass with diameters of about 3–300nm can be coated with molecular species that will enable them tocovalently bind selectively to a binding partner. For example, as shownin FIG. 9C, nanoparticles that are covalently coated with streptavidincan be used to enhance the visibility of biotin-tagged binding partnerson the biosensor surface. While a streptavidin molecule itself has amolecular weight of 60 kDa, the derivatized bead can have a molecularweight of any size, including, for example, 60 KDa. Binding of a largebead will result in a large change in the optical density upon thebiosensor surface, and an easily measurable signal. This method canresult in an approximately 1000× enhancement in sensitivity resolution.

Liquid-Containing Vessels

A grating of the invention can comprise an inner surface, for example, abottom surface of a liquid-containing vessel. A liquid-containing vesselcan be, for example, a microtiter plate well, a test tube, a petri dish,or a microfluidic channel. One embodiment of this invention is abiosensor that is incorporated into any type of microtiter plate. Forexample, a biosensor can be incorporated into the bottom surface of amicrotiter plate by assembling the walls of the reaction vessels overthe resonant reflection surface, as shown in FIGS. 32A and 32B, so thateach reaction “spot” can be exposed to a distinct test sample.Therefore, each individual microtiter plate well can act as a separatereaction vessel. Separate chemical reactions can, therefore, occurwithin adjacent wells without intermixing reaction fluids and chemicallydistinct test solutions can be applied to individual wells.

Several methods for attaching a biosensor or grating of the invention tothe bottom surface of bottomless microtiter plates can be used,including, for example, adhesive attachment, ultrasonic welding, andlaser welding.

The most common assay formats for pharmaceutical high-throughputscreening laboratories, molecular biology research laboratories, anddiagnostic assay laboratories are microtiter plates. The plates arestandard-sized plastic cartridges that can contain about 2, 6, 8, 24,48, 96, 384, 1536 or 3456 individual reaction vessels arranged in agrid. Due to the standard mechanical configuration of these plates,liquid dispensing, robotic plate handling, and detection systems aredesigned to work with this common format. A biosensor of the inventioncan be incorporated into the bottom surface of a standard microtiterplate. See, e g., FIG. 32A. Because the biosensor surface can befabricated in large areas, and because the readout system does not makephysical contact with the biosensor surface, an arbitrary number ofindividual biosensor areas can be defined that are only limited by thefocus resolution of the illumination optics and the x-y stage that scansthe illumination/detection probe across the biosensor surface.

Methods of Using Biosensors

Biosensors of the invention can be used to study one or a number ofspecific binding substance/binding partner interactions in parallel.Binding of one or more specific binding substances to their respectivebinding partners can be detected, without the use of labels, by applyingone or more binding partners to a biosensor that have one or morespecific binding substances immobilized on their surfaces. A biosensoris illuminated with light and a maxima in reflected wavelength, or aminima in transmitted wavelength of light is detected from thebiosensor. If one or more specific binding substances have bound totheir respective binding partners, then the reflected wavelength oflight is shifted as compared to a situation where one or more specificbinding substances have not bound to their respective binding partners.Where a biosensor is coated with an array of one or more distinctlocations containing the one or more specific binding substances, then amaxima in reflected wavelength or minima in transmitted wavelength oflight is detected from each distinct location of the biosensor.

In one embodiment of the invention, a variety of specific bindingsubstances, for example, antibodies, can be immobilized in an arrayformat onto a biosensor of the invention. See, e.g., FIG. 31. Thebiosensor is then contacted with a test sample of interest comprisingbinding partners, such as proteins. Only the proteins that specificallybind to the antibodies immobilized on the biosensor remain bound to thebiosensor. Such an approach is essentially a large-scale version of anenzyme-linked immunosorbent assay; however, the use of an enzyme orfluorescent label is not required. For high-throughput applications,biosensors can be arranged in an array of arrays, wherein severalbiosensors comprising an array of specific binding substances arearranged in an array. See, e.g., FIG. 33. Such an array of arrays canbe, for example, dipped into microtiter plate to perform many assays atone time. In another embodiment, a biosensor can occur on the tip of afiber probe for in vivo detection of biochemical substance. See, FIG.14.

The activity of an enzyme can be detected by applying one or moreenzymes to a biosensor to which one or more specific binding substanceshave been immobilized. The biosensor is washed and illuminated withlight. The reflected wavelength of light is detected from the biosensor.Where the one or more enzymes have altered the one or more specificbinding substances of the biosensor by enzymatic activity by, forexample, cleaving all or a portion of a specific binding substance fromthe surface of a biosensor the reflected wavelength of light is shifted.

Another embodiment of the invention is a method of detecting cleavage ofone or more entire specific binding substances from a surface of acolorimetric resonant reflectance optical biosensor. The method involvesimmobilizing one or more binding substances onto the surface of thecolorimetric resonant reflectance optical biosensor at a distinctlocation, detecting a PWV of the distinct location, applying one or morecleaving molecules, detecting a PWV of the distinct location andcomparing the initial PWV with the subsequent PWV. The cleavage of oneor more entire specific binding substances is detected, and a peakwavelength value (PWV) is a relative measure of the specific bindingsubstance that is bound to the biosensor. A cleaving molecule is amolecule that can cleave another molecule. For example, a cleavingmolecule can be an enzyme such as a proteases, lipases, nucleases,lyases, peptidases, hydrolases, ligases, kinases and phosphatases.

A colorimetric resonant reflectance optical biosensor can comprise aninternal surface of a microtiter well, a microtiter plate, a test tube,a petri dish or a microfluidic channel. Immobilization of the specificbinding substance can be affected via binding to, for example, thefollowing functional groups: a nickel group, an amine group, an aldehydegroup, an acid group, an alkane group, an alkene group, an alkyne group,an aromatic group, an alcohol group, an ether group, a ketone group, anester group, an amide group, an amino acid group, a nitro group, anitrile group, a carbohydrate group, a thiol group, an organic phosphategroup, a lipid group, a phospholipid group or a steroid group. Further,the specific binding substance is immobilized on the surface of thecolorimetric resonant reflectance optical biosensor via physicaladsorption, chemical binding, electrochemical binding, electrostaticbinding, hydrophobic binding or hydrophilic binding.

One or more specific binding substances can be arranged in an array ofone or more distinct locations on the surface of the biosensor. The oneor more distinct locations can define microarray spots of about 50–500microns, or about 150–200 microns in diameter.

The method described above, that of detecting cleavage of one or moreentire specific binding substances from a surface of a colorimetricresonant reflectance optical biosensor, can also comprise alternativesteps. One or more specific binding substances can be immobilized in oneor more distinct locations defining an array within a well of amicrotiter plate. The one or more distinct locations defining themicroarray can be located upon the surface of a colorimetric resonantreflectance optical biosensor, which, in turn, comprises an internalsurface of a well. A PWV is detected for one or more distinct locationswithin the well. One or more cleaving molecules are applied to the well.Detection of a PWV for one or more distinct locations within the well isperformed. The initial PWV and the subsequent PWV are compared. Thecleavage of one or more entire specific binding substances at the one ormore distinct locations within the well is detected. A peak wavelengthvalue (PWV) is a relative measure of the specific binding substance thatis bound to the biosensor.

Another embodiment of the invention provides a method of detecting howeffectively a molecule inhibits the activity of an enzyme or bindingpartner, i.e., “inhibition activity” of the molecule. In one embodiment,is adding one or more molecules suspected of having inhibition activityare added to a biosensor to which one or more specific bindingsubstances are attached, followed by the addition of one or more enzymesknown to act upon the specific binding substances. For example, aprotease, lipase, nuclease, lyase, peptidase, hydrolase, ligase, kinase,phosphatase, or any other type of enzyme that would produce a detectablechange in a specific binding substance. The enzyme can effect a specificbinding partner by, for example, cleaving substantially the entiresingle binding substance or a portion of the single binding substancefrom the biosensor. One or more binding partners known to bind to one ormore specific binding substances immobilized on the biosensor can alsobe added to the biosensor.

A molecule with no inhibition activity allows the enzyme activity tooccur unabated; a molecule with substantially complete inhibitionactivity halts the reaction substantially completely; and a moleculewith partial inhibition halts the reaction partially. Additionally, amolecule with no inhibition activity allows a binding partner to bind toits specific binding substance. A molecule with partial inhibitionallows partial or weak binding of the binding partner to its specificbinding substance partner. A molecule with inhibition activity inhibitsthe binding of the binding partner to its specific binding partner.Thus, the method provides a technique of detecting inhibition activityof one or more molecules against enzymes or binding partners.

Detecting a PWV of one or more distinct locations is followed byapplying one or more molecules suspected of having inhibition activityto the one or more distinct locations and applying one or more enzymesor binding partners to the distinct locations. The PWV of the one ormore distinct locations is detected and compared to the initial PWV.Alternatively, the one or more molecules suspected of having inhibitionactivity can be mixed with the one or more enzymes or binding partners,which, together, can be applied to the one or more distinct locations. Adecrease or increase in the initial PWV above in relation to thesubsequent PWV above is (1) a relative measure of the proportion ofbinding substance that is altered by the enzyme or the amount of bindingpartners bound to the biosensor from the biosensor surface or (2) ameasure of relative effectiveness of one or more molecules suspected ofhaving inhibition activity.

The method described above, that of detecting inhibition activity of oneor more molecules against enzymes or binding partners can also comprisealternative steps. For example, one or more specific binding substancescan be immobilized in one or more distinct locations defining an arraywithin a well of a microtiter plate or other liquid holding device. Theone or more distinct locations defining an array are located upon thesurface of a colorimetric resonant reflectance optical biosensor whichcomprises an internal surface of the well. Detecting a PWV for the oneor more distinct locations within the well is followed by applying oneor more molecules suspected of having inhibition activity to the well.One or more enzymes or binding partners are applied to the well and aPWV is detected for the one or more distinct locations within the well.The initial PWV is compared with the subsequent PWV and reveals theinhibition activity of one or more molecules against enzymes or bindingpartners at each distinct location within a well. Alternatively, the oneor more molecules suspected of having inhibition activity can be mixedwith the one or more enzymes or binding partners, which, together, canbe applied to the well.

Additionally, a test sample, for example, cell lysates containingbinding partners, can be applied to a biosensor of the invention,followed by washing to remove unbound material. The binding partnersthat bind to a biosensor can be eluted from the biosensor and identifiedby, for example, mass spectrometry. Optionally, a phage DNA displaylibrary can be applied to a biosensor of the invention followed bywashing to remove unbound material. Individual phage particles bound tothe biosensor can be isolated and the inserts in these phage particlescan then be sequenced to determine the identity of the binding partner.

Another embodiment of the invention provides a method of detecting cellmigration and chemotaxis. In particular, cells can be grown on one endof a colorimetric resonant reflectance optical biosensor, either withina well or in an array format. The end of the biosensor containing thecells can optionally be segregated, via the use of semi-permeablemembranes, from the opposing end where the chemotactic agent is placed.Detection systems comprised of an imaging spectrometer, or alternativelya fiber optic probe that can be moved to read from multiple locations ofthe biosensor, can then be used to detect the location of the cells, andin turn permit the computation of the cell migration velocity.

A further embodiment of the invention provides a method of detecting achange in cell growth patterns. Briefly, cells can be grown on acolorimetric resonant reflectance optical biosensor; a PWV detected; atest reagent applied to the cells; a PWV detected; and the initial PWVwith the subsequent PWV can be compared, wherein the difference betweenthe initial PWV in relation to the subsequent PWV indicates a change incell growth patterns. A difference in PWV correlates with a change in acell growth pattern.

The change in cell growth pattern can be selected from the groupconsisting of cell morphology, cell adhesion, cell migration, cellproliferation and cell death. One type of prokaryotic or eukaryoticcells or two or more types of eukaryotic or prokaryotic cells can begrown on the biosensor. The biosensor can comprise an internal surfaceof a vessel selected from the group consisting of a microtiter well,microtiter plate, test tube, petri dish and microfluidic channel.

A still further embodiment of the invention provides a method ofdetecting molecules released from cells grown in a semi-permeableinternal sleeve held in contact with a colorimetric resonant reflectanceoptical biosensor. The semi-permeable internal sleeve can be, forexample, a removable porous or non-removable porous insert that is heldin contact with or close to the surface of a biosensor, wherein thesleeve is permeable to molecules secreted from the cells cultured on itssurface and wherein the sleeve is impermeable to whole cells. A sleevecan fit into the wells of a microtiter plate or other vessel wherein abiosensor of the invention comprises an internal surface of the wells orother vessel.

This method can comprise the following steps: immobilizing one or morespecific binding substances onto a surface of the colorimetric resonantreflectance optical biosensor at one or more distinct locations;detecting a PWV of the one or more distinct locations; growing cells inthe semi-permeable internal sleeve held in contact with the colorimetricresonant reflectance optical biosensor at the one or more distinctlocations; detecting the PWV of the one or more distinct locations; andcomparing the initial PWV with the subsequent PWV. The binding ofmolecules released from cells grown in the semi-permeable internalsleeve held in contact with the colorimetric resonant reflectanceoptical biosensor to the one or more specific binding substances isdetected. Further, the initial PWV is a relative measure of the specificbinding substance that is bound to the biosensor, and the differencebetween the initial PWV in relation to the subsequent PWV is a relativemeasure of the molecules released from cells grown in a semi-permeableinternal sleeve that are bound to the specific binding substances. Thesemi-permeable internal sleeve is a removable porous or non-removableporous insert.

The method described above, that of detecting molecules released fromcells grown in a semi-permeable internal sleeve held in contact with acolorimetric resonant reflectance optical biosensor can also comprisealternative steps. For example, one or more binding substance can beimmobilized in one or more distinct locations defining an array within awell of a microtiter plate, wherein the colorimetric resonantreflectance optical biosensor comprises an internal surface of the well.Detecting a PWV for the one or more distinct locations defining an arraywithin the well is followed by growing cells in a semi-permeableinternal sleeve held in contact with the well. The final steps aredetecting the PWV for the one or more distinct locations within the welland comparing the initial PWV with the subsequent PWV. The differencebetween the initial PWV in relation to the subsequent PWV indicates therelative binding of one or more molecules secreted from the cellsgrowing on the semi-permeable internal sleeve within a well to the oneor more specific binding substances immobilized at one or more distinctlocations within the well on the surface of a colorimetric resonantreflectance optical biosensor.

The ability to detect the binding of binding partners to specificbinding substances, optionally followed by the ability to detect theremoval of substantially entire or partial bound specific bindingsubstances, from one or more distinct locations of the biosensor is animportant aspect of the invention. Biosensors of the invention are alsocapable of detecting and quantifying the amount of a binding partnerfrom a sample that is bound to one or more distinct locations definingan array by measuring the shift in reflected wavelength of light. Forexample, the wavelength shift at one or more distinct locations can becompared to positive and negative controls at other distinct locationsto determine the amount of a specific binding substance that is bound.Importantly, numerous such one or more distinct locations can bearranged on the biosensor surface, and the biosensor can comprise aninternal surface of a vessel such as an about 2, 6, 8, 24, 48, 96, 384,1536 or 3456 well-microtiter plate. As an example, where 96 biosensorsare attached to a holding fixture and each biosensor comprises about 100distinct locations, about 9600 biochemical assays can be performedsimultaneously.

Therefore, unlike methods for assays for surface plasmon resonance,resonant mirrors, and waveguide biosensors, the described methods enablemany thousands of individual binding reactions to take placesimultaneously upon the resonant optical biosensor surface. Clearly,this technology is useful in applications where large numbers ofbiomolecular interactions are measured in parallel, particularly whenmolecular labels will alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by this approach.

Detection Systems

A detection system can comprise a biosensor a light source that directslight to the biosensor, and a detector that detects light reflected fromthe biosensor. In one embodiment, it is possible to simplify the readoutinstrumentation by the application of a filter so that only positiveresults over a determined threshold trigger a detection.

A light source can illuminate a biosensor from its top surface, i.e.,the surface to which one or more specific binding substances areimmobilized or from its bottom surface. By measuring the shift inresonant wavelength at each distinct location of a biosensor of theinvention, it is possible to determine which distinct locations havebinding partners bound to them. The extent of the shift can be used todetermine the amount of binding partners in a test sample and thechemical affinity between one or more specific binding substances andthe binding partners of the test sample.

A biosensor can be illuminated twice. The first measurement determinesthe reflectance spectra of one or more distinct locations of a biosensorarray with one or more specific binding substances immobilized on thebiosensor. The second measurement determines the reflectance spectraafter one or more binding partners are applied to a biosensor. Thedifference in peak wavelength between these two measurements is ameasurement of the amount of binding partners that have specificallybound to a biosensor or one or more distinct locations of a biosensor.This method of illumination can control for small nonuniformities in asurface of a biosensor that can result in regions with slight variationsin the peak resonant wavelength. This method can also control forvarying concentrations or molecular weights of specific bindingsubstances immobilized on a biosensor.

Computer simulation can be used to determine the expected dependencebetween a peak resonance wavelength and an angle of incidentillumination. A biosensor as shown in FIG. 1 can be for purposes ofdemonstration. The substrate chosen was glass (n_(substrate)=1.50). Thegrating is an optical pattern of silicon nitride squares (t₂=180 nm,n₂=2.01 (n=refractive index), k₂=0.001 (k=absorption coefficient)) witha period of 510 nm, and a filling factor of 56.2% (i.e., 56.2% of thesurface is covered with silicon nitride squares while the rest is thearea between the squares). The areas between silicon nitride squares arefilled with a lower refractive index material. The same material alsocovers the squares and provides a uniformly flat upper surface. For thissimulation, a glass layer was selected (n₁=1.40) that covers the siliconnitride squares by t₂=100 nm.

The reflected intensity as a function of wavelength was modeled usingGSOLVER software, which utilizes full 3-dimensional vector code usinghybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination can be from any incidence and any polarization.

FIG. 10 plots the dependence of the peak resonant wavelength upon theincident illumination angle. The simulation shows that there is a strongcorrelation between the angle of incident light, and the peak wavelengththat is measured. This result implies that the collimation of theilluminating beam, and the alignment between the illuminating beam andthe reflected beam will directly affect the resonant peak linewidth thatis measured. If the collimation of the illuminating beam is poor, arange illuminating angles will be incident on the biosensor surface, anda wider resonant peak will be measured than if purely collimated lightwere incident.

Because the lower sensitivity limit of a biosensor is related to theability to determine the peak maxima, it is important to measure anarrow resonant peak. Therefore, the use of a collimating illuminationsystem with the biosensor provides for the highest possible sensitivity.

One type of detection system for illuminating the biosensor surface andfor collecting the reflected light is a probe containing, for example,six illuminating optical fibers that are connected to a light source,and a single collecting optical fiber connected to a spectrometer. Thenumber of fibers is not critical, any number of illuminating orcollecting fibers are possible. The fibers are arranged in a bundle sothat the collecting fiber is in the center of the bundle, and issurrounded by the six illuminating fibers. The tip of the fiber bundleis connected to a collimating lens that focuses the illumination ontothe surface of the biosensor.

In this probe arrangement, the illuminating and collecting fibers areside-by-side. Therefore, when the collimating lens is correctly adjustedto focus light onto the biosensor surface, one observes six clearlydefined circular regions of illumination, and a central dark region.Because the biosensor does not scatter light, but rather reflects acollimated beam, no light is incident upon the collecting fiber, and noresonant signal is observed. Only by defocusing the collimating lensuntil the six illumination regions overlap into the central region isany light reflected into the collecting fiber. Because only defocused,slightly uncollimated light can produce a signal, the biosensor is notilluminated with a single angle of incidence, but with a range ofincident angles. The range of incident angles results in a mixture ofresonant wavelengths due to the dependence shown in FIG. 10. Thus, widerresonant peaks are measured than might otherwise be possible.

Therefore, it is desirable for the illuminating and collecting fiberprobes to spatially share the same optical path. Several methods can beused to co-locate the illuminating and collecting optical paths. Forexample, a single illuminating fiber, which is connected at its firstend to a light source that directs light at the biosensor, and a singlecollecting fiber, which is connected at its first end to a detector thatdetects light reflected from the biosensor, can each be connected attheir second ends to a third fiber probe that can act as both anilluminator and a collector. The third fiber probe is oriented at anormal angle of incidence to the biosensor and supportscounter-propagating illuminating and reflecting optical signals. Anexample of such a detection system is shown in FIG. 11.

Another method of detection involves the use of a beam splitter thatenables a single illuminating fiber, which is connected to a lightsource, to be oriented at a 90 degree angle to a collecting fiber, whichis connected to a detector. Light is directed through the illuminatingfiber probe into the beam splitter, which directs light at thebiosensor. The reflected light is directed back into the beam splitter,which directs light into the collecting fiber probe. An example of sucha detection device is shown in FIG. 12. A beam splitter allows theilluminating light and the reflected light to share a common opticalpath between the beam splitter and the biosensor, so perfectlycollimated light can be used without defocusing.

Angular Scanning

Detection systems of the invention are based on collimated white lightillumination of a biosensor surface and optical spectroscopy measurementof the resonance peak of the reflected beam. Molecular binding on thesurface of a biosensor is indicated by a shift in the peak wavelengthvalue, while an increase in the wavelength corresponds to an increase inmolecular absorption.

As shown in theoretical modeling and experimental data, the resonancepeak wavelength is strongly dependent on the incident angle of thedetection light beam. FIG. 10 depicts this dependence as modeled for abiosensor of the invention. Because of the angular dependence of theresonance peak wavelength, the incident white light needs to be wellcollimated. Angular dispersion of the light beam broadens the resonancepeak, and reduces biosensor detection sensitivity. In addition, thesignal quality from the spectroscopic measurement depends on the powerof the light source and the sensitivity of the detector. In order toobtain a high signal-to-noise ratio, an excessively long integrationtime for each detection location can be required, thus lengtheningoverall time to readout a biosensor plate. A tunable laser source can beused for detection of grating resonance, but is expensive.

In one embodiment of the invention, these disadvantages are addressed byusing a laser beam for illumination of a biosensor, and a light detectorfor measurement of reflected beam power. A scanning mirror device can beused for varying the incident angle of the laser beam, and an opticalsystem is used for maintaining collimation of the incident laser beam.See, e.g., “Optical Scanning” (Gerald F. Marchall ed., Marcel Dekker(1991). Any type of laser scanning can be used. For example, a scanningdevice that can generate scan lines at a rate of about 2 lines to about1,000 lines per second is useful in the invention. In one embodiment ofthe invention, a scanning device scans from about 50 lines to about 300lines per second.

In one embodiment, the reflected light beam passes through part of thelaser scanning optical system, and is measured by a single lightdetector. The laser source can be a diode laser with a wavelength of,for example, 780 nm, 785 nm, 810 nm, or 830 nm. Laser diodes such asthese are readily available at power levels up to 150 mW, and theirwavelengths correspond to high sensitivity of Si photodiodes. Thedetector thus can be based on photodiode biosensors. An example of sucha detection system is shown in FIG. 13. A light source (300) provideslight to a scanner device (400), which directs the light into an opticalsystem (500). The optical system (500) directs light to a biosensor(600). Light is reflected from the biosensor (600) to the optical system(500), which then directs the light into a light signal detector (700).One embodiment of a detection system is shown in FIG. 30, whichdemonstrates that while the scanning mirror changes its angularposition, the incident angle of the laser beam on the surface changes bynominally twice the mirror angular displacement. The scanning mirrordevice can be a linear galvanometer, operating at a frequency of about 2Hz up to about 120 Hz, and mechanical scan angle of about 10 degrees toabout 20 degrees. In this example, a single scan can be completed withinabout 10 msec. A resonant galvanometer or a polygon scanner can also beused. The example shown in FIG. 30 includes a simple optical system forangular scanning. It consists of a pair of lenses with a common focalpoint between them. The optical system can be designed to achieveoptimized performance for laser collimation and collection of reflectedlight beam.

The angular resolution depends on the galvanometer specification, andreflected light sampling frequency. Assuming galvanometer resolution of30 arcsec mechanical, corresponding resolution for biosensor angularscan is 60 arcsec, i.e. 0.017 degree. In addition, assume a samplingrate of 100 ksamples/sec, and 20 degrees scan within 10 msec. As aresult, the quantization step is 20 degrees for 1000 samples, i.e. 0.02degree per sample. In this example, a resonance peak width of 0.2degree, as shown by Peng and Morris (Experimental demonstration ofresonant anomalies in diffraction from two-dimensional gratings, OpticsLett., 21:549 (1996)), will be covered by 10 data points, each of whichcorresponds to resolution of the detection system.

The advantages of such a detection system includes: excellentcollimation of incident light by a laser beam, high signal-to-noiseratio due to high beam power of a laser diode, low cost due to a singleelement light detector instead of a spectrometer, and high resolution ofresonance peak due to angular scanning.

Fiber Probe Biosensor

A biosensor of the invention can occur on the tip of a multi-mode fiberoptic probe. This fiber optic probe allows for in vivo detection ofbiomarkers for diseases and conditions, such as, for example, cardiacartery disease, cancer, inflammation, and sepsis. A single biosensorelement (comprising, for example, several hundred grating periods) canbe fabricated into the tip of a fiber optic probe, or fabricated from aglass substrate and attached to the tip of a fiber optic probe. See FIG.14. A single fiber is used to provide illumination and measure resonantreflected signal.

For example, a fiber probe structure similar to that shown in FIG. 11can be used to couple an illuminating fiber and detecting fiber into asingle counterpropagating fiber with a biosensor embedded or attached toits tip. The fiber optic probe is inserted into a mammalian body, forexample, a human body. Illumination and detection of a reflected signalcan occur while the probe is inserted in the body.

The following are provided for exemplification purpose only and are notintended to limit the scope of the invention described in broad termsabove. All references cited in this disclosure are incorporated hereinby reference.

EXAMPLE 1

Immobilized Protein Detection

In order to demonstrate a biosensor's ability to quantify biomoleculeson its surface, droplets of BSA dissolved in H₂O at variousconcentrations were applied to a biosensor as shown in FIG. 1. The 3 μldroplets were allowed to dry in air, leaving a small quantity of BSAdistributed over a ˜2 mm diameter area. The peak resonant wavelength ofeach biosensor location was measured before and after dropletdeposition, and the peak wavelength shift was recorded. See FIG. 34.

EXAMPLE 2

Immobilization of One or More Specific Binding Substances

The following protocol was used on a colorimetric resonant reflectivebiosensor to activate the surface with amine functional groups. Aminegroups can be used as a general-purpose surface for subsequent covalentbinding of several types of linker molecules.

A glass substrate biosensor of the invention is cleaned by immersing itinto piranha etch (70/30% (v/v) concentrated sulfuric acid/30% hydrogenperoxide) for 12 hours. The biosensor was washed thoroughly with water.The biosensor was dipped in 3% 3-aminopropyltriethoxysilane solution indry acetone for 1 minute and then rinsed with dry acetone and air-dried.Alternatively, immersion of the biosensor in 10%3-aminopropyltriethoxysilane (Pierce) solution in ethanol (Aldrich) for1 min, followed by a brief ethanol rinse. Activated sensors were thendried at 70° C. for 10 minutes. The biosensor was then washed withwater.

A semi-quantitative method is used to verify the presence of aminogroups on the biosensor surface. One biosensor from each batch ofamino-functionalized biosensors is washed briefly with 5 mL of 50 mMsodium bicarbonate, pH 8.5. The biosensor is then dipped in 5 mL of 50mM sodium bicarbonate, pH 8.5 containing 0.1 mMsulfo-succinimidyl-4-O-(4,4′-dimethoxytrityl)-butyrate (s-SDTB, Pierce,Rockford, Ill.) and shaken vigorously for 30 minutes. The s-SDTBsolution is prepared by dissolving 3.0 mg of s-SDTB in 1 mL of DMF anddiluting to 50 mL with 50 mM sodium bicarbonate, pH 8.5. After a 30minute incubation, the biosensor is washed three times with 20 mL ofddH₂O and subsequently treated with 5 mL 30% perchloric acid. Thedevelopment of an orange-colored solution indicates that the biosensorhas been successfully derivatized with amines; no color change isobserved for untreated glass biosensors.

The absorbance at 495 nm of the solution after perchloric acid treatmentfollowing the above procedure can be used as an indicator of thequantity of amine groups on the surface. In one set of experiment, theabsorbance was 0.627, 0.647, and 0.728 for Sigma slides, Cel-Associateslides, and in-house biosensor slides, respectively. This indicates thatthe level of NH₂ activation of the biosensor surface is comparable inthe activation commercially available microarray glass slides.

After following the above protocol for activating the biosensor withamine, a linker molecule can be attached to the biosensor. Whenselecting a cross-linking reagent, issues such as selectivity of thereactive groups, spacer arm length, solubility, and cleavability shouldbe considered. The linker molecule, in turn, binds the specific bindingsubstance that is used for specific recognition of a binding partner. Asan example, the protocol below has been used to bind a biotin linkermolecule to the amine-activated biosensor.

Protocol for Activating Amine-Coated Biosensor with Biotin

Wash an amine-coated biosensor with PBS (pH 8.0) three times. Preparesulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin,Pierce, Rockford, Ill.) solution in PBS buffer (pH 8) at 0.5 mg/mlconcentration. Add 2 ml of the sulfo-NHS-LC-biotin solution to eachamine-coated biosensor and incubate at room temperature for 30 min. Washthe biosensor three times with PBS (pH 8.0). The sulfo-NHS-LC-biotinlinker has a molecular weight of 556.58 and a length of 22.4 Å. Theresulting biosensors can be used for capturing avidin or strepavidinmolecules.

Protocol for Activating Amine-Coated Biosensor with Aldehyde

Prepare 2.5% glutaraldehyde solution in 0.1 M sodium phosphate, 0.05%sodium azide, 0.1% sodium cyanoborohydride, pH 7.0. Add 2 ml of thesulfo-NHS-LC-biotin solution to each amine-coated biosensor and incubateat room temperature for 30 min. Wash the biosensor three times with PBS(pH 7.0). The glutaraldehyde linker has a molecular weight of 100.11.The resulting biosensors can be used for binding proteins and otheramine-containing molecules. The reaction proceeds through the formationof Schiff bases, and subsequent reductive amination yields stablesecondary amine linkages. In one experiment, where a coated aldehydeslide made by the inventors was compared to a commercially availablealdehyde slide (Cel-Associate), ten times higher binding of streptavidinand anti-rabbit IgG on the slide made by the inventors was observed.

Protocol for Activating Amine-coated Biosensor with NHS

25 mM N,N′-disuccinimidyl carbonate (DSC, Sigma Chemical Company, St.Louis, Mo.) in sodium carbonate buffer (pH 8.5) was prepared. 2 ml ofthe DSC solution was added to each amine-coated biosensor and incubatedat room temperature for 2 hours. The biosensors were washed three timeswith PBS (pH 8.5). A DSC linker has a molecular weight of 256.17.Resulting biosensors are used for binding to hydroxyl- oramine-containing molecules. This linker is one of the smallesthomobifunctional NHS ester cross-linking reagents available.

In addition to the protocols defined above, many additional surfaceactivation and molecular linker techniques have been reported thatoptimize assay performance for different types of biomolecules. Mostcommon of these are amine surfaces, aldehyde surfaces, and nickelsurfaces. The activated surfaces, in turn, can be used to attach severaldifferent types of chemical linkers to the biosensor surface, as shownin Table 2. While the amine surface is used to attach several types oflinker molecules, the aldehyde surface is used to bind proteinsdirectly, without an additional linker. A nickel surface is usedexclusively to bind molecules that have an incorporated histidine(“his”) tag. Detection of “his-tagged” molecules with a Nickel activatedsurface is well known (Sigal et al., Anal. Chem. 68, 490 (1996)). Table2 demonstrates an example of the sequence of steps that are used toprepare and use a biosensor, and various options that are available forsurface activation chemistry, chemical linker molecules, specificbinding substances and binding partners molecules. Opportunities alsoexist for enhancing detected signal through amplification with largermolecules such as HRP or streptavidin and the use of polymer materialssuch as dextran or TSPS to increase surface area available for molecularbinding.

TABLE 2 Label Bare Surface Linker Receptor Detected Molecule SensorActivation Molecule Molecule Material (Optional) Glass Amine SMPT Smm'cules Peptide Enhance Polymers Aldehyde NHS-Biotin Peptide Med Proteinsensitivity optional to Ni DMP Med Protein Lrg Protein 1000x enhanceNNDC Lrg Protein IgG HRP sensitivity His-tag .IgG Phage Streptavidin2–5x Others. . . cDNA Cell Dextran cDNA TSPS

EXAMPLE 3

IgG Assay

As an initial demonstration for detection of biochemical binding, anassay was performed in which a biosensor was prepared by activation withthe amino surface chemistry described in Example 2 followed byattachment of a biotin linker molecule. The biotin linker is used tospecifically interact with and effectively tether a streptavidinreceptor molecule to the surface by exposure to a 50 μg/ml concentrationsolution of streptavidin in PBS at room temperature for 2–4 hours. Thestreptavidin receptor is capable of binding any biotinylated protein tothe biosensor surface. For this example, 3 μl droplets of biotinylatedanti-human IgG in phosphate buffer solution (PBS) were deposited onto 4separate locations on the biosensor surface at a concentration of 200μg/ml. The solution was allowed to incubate on the biosensor for 30minutes before rinsing thoroughly with PBS. The peak resonant wavelengthof the 4 locations were measured after biotin activation, afterstreptavidin receptor application, and after ah-IgG binding. FIG. 34shows that the addition of streptavidin and ah-IgG both yield a clearlymeasurable increase in the resonant wavelength.

EXAMPLE 4

Biotin/Streptavidin Assay

A series of assays were performed to detect streptavidin binding by abiotin receptor layer. A biosensor was first activated with aminechemistry, followed by attachment of a NHS-Biotin linker layer, aspreviously described. Next, 3 μl droplets of streptavidin in PBS wereapplied to the biosensor at various concentrations. The droplets wereallowed to incubate on the biosensor surface for 30 min beforethoroughly washing with PBS rinsing with DI water. The peak resonantwavelength was measured before and after streptavidin binding, and theresonant wavelength shifts are shown in FIG. 16. A linear relationshipbetween peak wavelength and streptavidin concentration was observed, andin this case the lowest streptavidin concentration measured was 0.2μg/ml. This concentration corresponds to a molarity of 3.3 nM.

EXAMPLE 5

Protein-Protein Binding Assay

An assay was performed to demonstrate detection of protein-proteininteractions. As described previously, a biosensor was activated withamine chemistry and an NHS-biotin linker layer. A goat anti-biotinantibody receptor layer was attached to the biotin linker by exposingthe biosensor to a 50 μg/ml concentration solution in PBS for 60 min atroom temperature followed by washing in PBS and rinsing with DI water.In order to prevent interaction of nonspecific proteins with unboundbiotin on the biosensor surface, the biosensor surface was exposed to a1% solution of bovine serum albumin (BSA) in PBS for 30 min. The intentof this step is to “block” unwanted proteins from interacting with thebiosensor. As shown in FIG. 17 a significant amount of BSA isincorporated into the receptor layer, as shown by the increase in peakwavelength that is induced. Following blocking, 3 μl droplets of variousconcentrations of anti-goat IgG were applied to separate locations onthe biosensor surface. The droplets were allowed to incubate for 30 minbefore thorough rinsing with DI water. The biosensor peak resonantwavelength was measured before blocking, after blocking, after receptorlayer binding, and after anti-goat IgG detection for each spot. FIG. 17shows that an anti-goat IgG concentration of 10 μg/ml yields an easilymeasurable wavelength shift.

EXAMPLE 6

Unlabeled ELISA Assay

Another application of a biosensor array platform is its ability toperform Enzyme-Linked Immunosorbent Assays (ELISA) without the need foran enzyme label, and subsequent interaction an enzyme-specific substrateto generate a colored dye. FIG. 18 shows the results of an experimentwhere a biosensor was prepared to detect interferon-γ (IFN-γ) with anIFN-γ antibody receptor molecule. The receptor molecule was covalentlyattached to an NH₂-activated biosensor surface with an SMPT linkermolecule (Pierce Chemical Company, Rockford, Ill.). The peak resonantwavelength shift for application of the NH₂, SMPT, and anti-human IFN-γreceptor molecules were measured for two adjacent locations on thebiosensor surface, as shown in FIG. 18. The two locations were exposedto two different protein solutions in PBS at a concentration of 100μg/ml. The first location was exposed to IFN-γ, which is expected tobind with the receptor molecule, while the second was exposed to neuralgrowth factor (NGF), which is not expected to bind with the receptor.Following a 30 minute incubation the biosensor was measured byilluminating from the bottom, while the top surface remained immersed inliquid. The location exposed to IFN-γ registered a wavelength shift of0.29 nm, while the location exposed to NGF registered a wavelength shiftof only 0.14 nm. Therefore, without the use of any type of enzyme labelor color-generating enzyme reaction, the biosensor was able todiscriminate between solutions containing different types of protein.

EXAMPLE 7

Protease Inhibitor Assay (Caspase-3)

A Caspase-3 protease inhibitor assay was performed to demonstrate thebiosensor's ability to measure the presence and cleavage of smallmolecules in an experimental context that is relevant to pharmaceuticalcompound screening.

Caspases (Cysteine-requiring Aspartate protease) are a family ofproteases that mediate cell death and are important in the process ofapoptosis. Caspase 3, an effector caspase, is the most studied ofmammalian caspases because it can specifically cleave most knowncaspase-related substrates. The caspase 3 assay is based on thehydrolysis of the 4-amino acid peptide substrate NHS-Gly-Asp-Glu-Val-Aspp-nitroanilide (NHS-GDEVD-pNA) by caspase 3, resulting in the release ofthe pNA moiety.

The NHS molecule attached to the N-terminal of the GDEVD provides areactive end group to enable the NHS-GDEVD-pNA complex to be covalentlybound to the biosensor with the pNA portion of the complex oriented awayfrom the surface. Attached in this way, the caspase-3 will have the bestaccess to its substrate cleavage site.

A biosensor was prepared by cleaning in 3:1 H₂SO₄:H₂O₂ solution (roomtemperature, 1 hour), followed by silanation (2% silane in dry acetone,30 sec) and attachment of a poly-phe-lysine (PPL) layer (100 μg/ml PPLin PBS pH 6.0 with 0.5 M NaCl, 10 hours). The NHS-GDEVD-pNA complex wasattached by exposing the biosensor to a 10 mM solution in PBS (pH 8.0,room temperature, 1 hour). A microwell chamber was sealed over thebiosensor surface, and cleavage of pNA was performed by addition of 100μl of caspase-3 in 1× enzyme buffer (100 ng/ml, room temperature, 90minutes). Following exposure to the caspase 3 solution, the biosensorwas washed in PBS. A separate set of experiments using aspectrophotometer were used to confirm the attachment of the complex tothe surface of the biosensor, and functional activity of the caspase-3for removal of the pNA molecule from the surface-bound complex.

The peak resonant frequency of the biosensor was measured beforeattachment of the NHS-GDEVD-pNA complex, after attachment of the complex(MW=860 Da), and after cleavage of the pNA (MW=136) with caspase 3. Asshown in FIG. 19, the attachment of the peptide molecule is clearlymeasurable, as is the subsequent removal of the pNA. The pNA removalsignal of Δλ=0.016 nm is 5.3× higher than the minimum detectable peakwavelength shift of 0.003 nm. The proportion of the added molecularweight and subtracted molecular weight (860 Da/136 Da=6.32) are in closeagreement with the proportion of peak wavelength shift observed for theadded and subtracted material (0.082 nm/0.016 nm=5.14).

The results of this experiment confirm that a biosensor is capable ofmeasuring small peptides (in this case, a 5-mer peptide) without labels,and even detecting the removal of 130 Da portions of a molecule throughthe activity of an enzyme.

EXAMPLE 8

Reaction Kinetics for Protein-Protein Binding Assays

Because a biosensor of the invention can be queried continuously as afunction of time while it is immersed in liquid, a biosensor can beutilized to perform both endpoint-detection experiments and to obtainkinetic information about biochemical reactions. As an example, FIG. 20shows the results of an experiment in which a single biosensor locationis measured continuously through the course of consecutively addingvarious binding partners to the surface. Throughout the experiment, adetection probe illuminated the biosensor through the back of thebiosensor substrate, while biochemistry is performed on the top surfaceof the device. A rubber gasket was sealed around the measured biosensorlocation so that added reagents would be confined, and all measurementswere performed while the top surface of the biosensor was immersed inbuffer solution. After initial cleaning, the biosensor was activatedwith NH₂, and an NHS-Biotin linker molecule. As shown in FIG. 20, goatα-biotin antibodies of several different concentrations (1, 10, 100,1000 μg/ml) were consecutively added to the biosensor and allowed toincubate for 30 minutes while the peak resonant wavelength wasmonitored. Following application of the highest concentration α-BiotinIgG, a second layer of protein was bound to the biosensor surfacethrough the addition of α-goat IgG at several concentrations (0.1, 1,10, and 100 μg/ml). Again, the resonant peak was continuously monitoredas each solution was allowed to incubate on the biosensor for 30minutes. FIG. 20 shows how the resonant peak shifted to greaterwavelength at the end of each incubation period.

FIG. 21 shows the kinetic binding curve for the final resonant peaktransitions from FIG. 20, in which 100 μg/ml of α-goat IgG is added tothe biosensor. The curve displays the type of profile that is typicallyobserved for kinetic binding experiments, in which a rapid increase fromthe base frequency is initially observed, followed by a gradualsaturation of the response. This type of reaction profile was observedfor all the transitions measured in the experiment. FIG. 22 shows thekinetic binding measurement of IgG binding.

The removal of material from the biosensor surface through the activityof an enzyme is also easily observed. When the biosensor from the aboveexperiment (with two protein coatings of goat anti-biotin IgG andanti-goat IgG) is exposed to the protease pepsin at a concentration of 1mg/ml, the enzyme dissociates both IgG molecules, and removes them fromthe biosensor surface. As shown in FIG. 23, the removal of boundmolecules from the surface can be observed as a function of time.

EXAMPLE 9

Proteomics Applications

Biosensors of the invention can be used for proteomics applications. Abiosensor array can be exposed to a test sample that contains a mixtureof binding partners comprising, for example, proteins or a phage displaylibrary, and then the biosensor surface is rinsed to remove all unboundmaterial. The biosensor is optically probed to determine which distinctlocations on the biosensor surface have experienced the greatest degreeof binding, and to provide a quantitative measure of bound material.Next, the biosensor is placed in a “flow cell” that allows a small(e.g., <50 microliters) fixed volume of fluid to make contact to thebiosensor surface. One electrode is activated so as to elute boundmaterial from only selected biosensor array distinct locations. Thebound material becomes diluted within the flow cell liquid. The flowcell liquid is pumped away from the biosensor surface and is storedwithin a microtiter plate or some other container. The flow cell liquidis replaced with fresh solution, and a new biosensor electrode isactivated to elute its bound binding partners. The process is repeateduntil all biosensor distinct locations of interest have been eluted andgathered into separate containers. If the test sample liquid contained amixture of proteins, protein contents within the separate containers canbe analyzed using a technique such as electrospray tandem massspectrometry. If the sample liquid contained a phage display library,the phage clones within the separate containers can be identifiedthrough incubation with a host strain bacteria, concentrationamplification, and analysis of the relevant library DNA sequence.

EXAMPLE 10

Homogenous Assay Demonstration

An SWS biosensor detects optical density of homogenous fluids that arein contact with its surface, and is able to differentiate fluids withrefractive indices that differ by as little as Δn=4×10⁻⁵. Because asolution containing two free non-interacting proteins has a refractiveindex that is different from a solution containing two bound interactingproteins, an SWS biosensor can measure when a protein-proteininteraction has occurred in solution without any kind of particle tag orchemical label.

Three test solutions were prepared for comparison:

-   1. Avidin in Phosphate Buffer Solution (PBS), (10 μg/ml)-   2. Avidin (10 μg/ml)+Bovine Serum Albumin (BSA) (10 μg/ml) in PBS-   3. Avidin (10 μg/ml)+Biotinylated BSA (b−BSA) (10 μg/ml) in PBS    A single SWS biosensor was used for all measurements to eliminate    any possibility of cross-biosensor bias. A 200 μl sample of each    test solution was applied to the biosensor and allowed to    equilibrate for 10 minutes before measurement of the SWS biosensor    peak resonant wavelength value. Between samples, the biosensor was    thoroughly washed with PBS.

The peak resonant wavelength values for the test solutions are plottedin FIG. 24. The avidin solution was taken as the baseline reference forcomparison to the Avidin+BSA and Avidin+b−BSA solutions. Addition of BSAto avidin results in only a small resonant wavelength increase, as thetwo proteins are not expected to interact. However, because biotin andavidin bind strongly (Kd=10⁻¹⁵M), the avidin+b−BSA solution will containlarger bound protein complexes. The peak resonant wavelength value ofthe avidin+b−BSA solution thus provides a large shift compared toavidin+BSA.

The difference in molecular weight between BSA (MW=66 KDa) and b−BSA(MW=68 KDa) is extremely small. Therefore, the differences measuredbetween a solution containing non-interacting proteins (avidin+BSA) andinteracting proteins (avidin+b−BSA) are attributable only to differencesin binding interaction between the two molecules. The bound molecularcomplex results in a solution with a different optical refractive indexthan the solution without bound complex. The optical refractive indexchange is measured by the SWS biosensor.

EXAMPLE 11

Microtiter Plate Assay

As a demonstration of the detection of biochemical binding on acolorimetric resonant reflectance optical biosensor which comprises aninternal surface of a microtiter plate, the following assay wasperformed. The protein-protein system selected for this study wasdetection of anti-biotin IgG antibody using biotin immobilized on thebiosensor surface as a specific binding substance. Therefore, a protocolfor immobilization of biotin on the biosensor surface was developed thatutilizes a bifunctional polyethyleneglycol-N-hydrosuccinimide (NHS-PEG)linker molecule (Shearwater Polymers, Inc.) to act as an intermediarybetween the amine surface group and the biotin. The NHS-PEG molecule isdesigned specifically to enable NHS to preferentially bind to theamine-activated surface, leaving the PEG portion of the moleculeoriented away from the surface. The NHS-PEG linker molecule serves toseparate the biotin molecule from the biosensor surface by a shortdistance so it may retain its conformation, and thus its affinity forother molecules. The PEG also serves to prevent nonspecific binding ofproteins to the biosensor.

After attachment of amine-activated biosensor sheets into the bottom ofmicrotiter plates, individual microtiter wells were prepared with threedifferent surface functional groups in order to provide sufficientexperimental controls for the detection of anti-biotin IgG. First,amine-activated surfaces were studied without additional modification.The amine-activated surface is expected to bind proteinsnonspecifically, but not with high affinity. Second, microtiter wellswith the NHS-PEG bifunctional linker molecule were prepared. The NHS-PEGmolecule is expected to provide a surface that does not bind protein.Third, microtiter wells with an NHS-PEG-Biotin linker molecule wereprepared. The NHS-PEG-Biotin molecule is expected to bind strongly toanti-biotin IgG.

To activate an amine-coated biosensor with biotin, 2 ml ofNHS-PEG-Biotin (Shearwater) solution in TPBS (a reference buffersolution of 0.01% TWEEN™ 20 in phosphate buffer solution, pH 8) at 1.0mg/ml concentration was added to the biosensor surface, and incubated at37° C. for 1 hour. An identical procedure was used for attachment of aNHS-PEG (Shearwater) molecule without biotin. All purchased reagentswere used as packaged.

A protein-antibody affinity assay was performed to demonstrate operationof a biosensor. A matrix of three separate biosensor surface states(NH₂, NHS-PEG, NHS-PEG-Biotin) were prepared and exposed to 7concentrations of goat anti-biotin IgG (Sigma). Each matrix location wasmeasured within a separate microtiter plate well, for a total of 21wells measured simultaneously. Because the NHS-PEG wells are notexpected to bind protein, they provide a reference for canceling commonmode effects such as the effect of the refractive index of the testsample and environmental temperature variation during the course of anassay.

FIG. 25 plots the PWV shift-referenced to a biosensor with no chemicalfunctional groups immobilized, recorded due to attachment of NH₂,NH₂+(NHS-PEG), and NH₂+(NHS-PEG-Biotin) molecules to the biosensorsurface. The error bars indicate the standard deviation of the recordedPWV shift over 7 microtiter plate wells. The data indicates that thebiosensor can differentiate between a clean surface, and one withimmobilized NH₂, as well as clearly detecting the addition of theNHS-PEG (MW=2000 Da) molecule. The difference between surfaceimmobilized NHS-PEG and NHS-PEG-Biotin (MW=3400 Da) is also measurable.

FIG. 26A-C shows the PWV shift response as a function of time for thebiosensor wells when exposed to various concentrations of anti-biotinIgG (0–80 mg/ml) and allowed to incubate for 20 minutes. The NHS-PEGsurface (FIG. 26B) provides the lowest response, while theamine-activated surface (FIG. 26A) demonstrates a low level ofnonspecific interaction with the anti-biotin IgG at high concentrations.The NHS-PEG-Biotin surface (FIG. 26C) clearly demonstrates strongspecific interaction with the anti-biotin IgG—providing strong PWVshifts in proportion to the concentration of exposed anti-biotin IgG.

The PWV shift magnitudes after 20 minutes from FIG. 26C are plotted as afunction of anti-biotin IgG concentration in FIG. 25. A roughly linearcorrelation between the IgG concentration and the measured PWV shift isobserved, and the lowest concentration IgG solution (1.25 μg/ml, 8.33nM) is clearly measurable over the negative control PSB solution.

The removal of material from the biosensor surface through the activityof an enzyme is shown in FIG. 27. When the biosensor is exposed to theprotease pepsin at a concentration of 1 mg/ml (volume=100 μl), theenzyme disassociates both goat-anti biotin IgG and anti-goat IgG andremoves them from the biosensor surface. The removal of bound moleculesfor the surface can be observed as a function of time.

EXAMPLE 12

Growing Cells on the Colorimetric Resonant Reflectance Optical Biosensor(96 Well Microtiter Plate)

A colorimetric resonant reflectance optical biosensor which comprises aninternal surface of a microtiter plate was sterilized prior to seedingof the culture. Sterilization was accomplished by placing the biosensorin Biosafety hood and exposing the microtiter plate and protection boxto UV light for 12 to 48 hours, more preferably about 16 to 36 hours,still more preferably 18 to 30 hours and most preferably about 24 hours.Cells were harvested from a living cultures of chondrocyte cells grownin chondrocyte growth medium (Cell Applications, Inc.) and HEK humankidney tumor cells (ATCC) grown in RPMI (Sigma). 1×10⁵ to 1×10⁶ cellswere added to each 96 well, the microtiter plate was placed into theprotection box and incubated in CO₂ incubator at 37° C. for 24–48 hours.

Cell growth at a biosensor location can be detected via the peakwavelength values of the colorimetric resonant reflectance opticalbiosensor surface or monitored more generally using a microscope,digital camera, conventional camera, or other visualization apparatus,magnifying or nonmagnifying, that utilizes lens-based optics orelectronics-based charge coupled device (CCD) technology.

EXAMPLE 13

Detect Cell Morphology Change on the Colorimetric Resonant ReflectanceOptical Biosensor

Cell morphology changes were detected utilizing a colorimetric resonantreflectance optical biosensor which comprises an internal surface of amicrotiter plate. Chondrocyte cells were grown to 10–90% confluentmonolayer according to Example 12. The cell monolayer was washed withsalt-balanced buffer such as Hank's medium (Sigma), and monolayerstability was tested washing or incubating the cells with thesalt-balanced medium such as Hank's medium for 10 minutes. Monolayerstability at any biosensor location can be assessed by detecting thepeak wavelength value at any location before, during or after thewashing or incubation step.

0.25% trypsin in Tris-EDTA was warmed to room temperature and added tothe biosensor wells while peak wavelength values were being detected.Blank controls were also utilized in which no cells were grown. As canbe seen in FIG. 28, reaction progress was followed over 12 minutes. Thecells were observed to remain attached to the surface of the biosensorthroughout the assay; the observed decrease in PWV upon addition of 2mg/ml trypsin to the biosensor wells containing the chondrocyte cellsindicates a change in chondrocyte cell morphology. The control (nochondrocyte cell) wells exhibited an insignificant response to theaddition of trypsin.

FIG. 29 shows the results of a cell adhesion assay using kidney tumorcells. Trypsin was added to six biosensor wells containing kidney tumorcells grown on the surface of the biosensor. Two wells were utilized asreplicate samples for each of the three trypsin concentrations. Uponaddition of the trypsin, a decrease in PWV is observed indicating thedetachment of the cells from the surface of the biosensor.

Both primary and tumor cell lines have been observed to grow well on thecolorimetric resonant reflectance optical biosensor surface within themicrotiter plate well, and eukaryotic cells have been observed togenerate very stable PWVs.

EXAMPLE 14

Detecting Molecules Released from Cells Grown in a Semi-PermeableInternal Sleeve Held in Contact with a Colorimetric Resonant ReflectanceOptical Biosensor

Molecules secreted, shed or otherwise ejected from cells can be detectedutilizing a colorimetric resonant reflectance optical biosensor whichcomprises an internal surface of a microtiter plate well. Antibodiesagainst interleukin-1 were immobilized on the surface of thecolorimetric resonant reflectance optical biosensor within themicrotiter plate well. A semi-permeable internal sleeve was insertedinto the well, followed by mouse macrophage cells (ATCC CRL-2019) andRPMI 1640 growth medium (Sigma). The cells were grown according to themethod of Example 12 while detecting the colorimetric resonantreflectance optical PWV at several locations on the biosensor surface.When lipopolysaccharide (Sigma) was then used to stimulate theproduction of interleukin-1, PWVs were seen to increase over time asinterleukin-1, secreted from the macrophage cells, diffused through thesemi-permeable internal sleeve and bound to the interleukin-1 antibodyimmobilized on the biosensor surface.

EXAMPLE 15

Protein Microarray Demonstration on the Colorimetric ResonantReflectance Optical Biosensor

The colorimetric resonant reflectance optical biosensor is able toperform assays in an array format; this example illustrates the abilityto detect differential binding affinities among different types of IgG.

In this example, a biosensor sheet was cut into a 1-in×2-in rectangularregion. The sensor is comprised of a hydrophobic TaO surface, to which 1mg/ml each of rabbit-IgG, chicken-IgG, goat-IgG, and human-IgG (all IgGfrom Sigma) are spotted using an Affymetrix GMS pin-and-loop spotterwith 400-μm spotting loops. In total, 4 rows of 7 spots each are made(see FIG. 35-A), in which the rabbit-IgG comprised the first row; thechicken-IgG comprised the second row; the goat-IgG comprised the thirdrow, and the human IgG comprised the fourth row. After spotting, theIgG's are incubated at room temperature for 30-min. Subsequently, a SRUMicroarray Scanner comprised of a Jobin Yvon high resolution imagingspectrometer is used to scan the resulting data, shown in FIG. 35-A.

After spotting, 1 mg/ml of gelatin (Sigma) is flowed over the surface asa blocking agent to prevent the binding agent (anti-human-IgG) fromnon-specifically binding to the non-spotted areas. The entire microarrayslide is then rinsed three times in PBS for 10-seconds each time,followed by three rinses in H₂O, also for 10-seconds each time. Theresulting microarray is then scanned using the SRU Microarray Scanner.Lastly, 100 μg/ml of anti-human-IgG is flowed over the entire surface,and allowed to incubate at room temperature for 30-min prior to rinsingusing the same PBS rinse procedure identified above. The resultingmicroarray containing the binding interactions is scanned using a SRUMicroarray Scanner once again. The difference between this final scanand the scan performed after blocking shows the amount of material boundto each spot on the microarray (see FIG. 35-B). In particular, the highdegree of affinity between the human-IgG and the anti-human-IgG isevident in the strong response corresponding to the bottom-most row ofFIG. 35-B. Additionally, at an intensity scale of 0.04 nm/intensitycount in FIG. 35-B, the binding result of 0.8-nm to 1.0-nm wavelengthshift observed by the microarray system is consistent to observationsmade in well-based assays.

EXAMPLE 16

DNA-DNA Binding Interaction Demonstration on the Colorimetric ResonantReflectance Optical Biosensor

To demonstrate the detection of hybridization events on the colorimetricresonant reflectance optical biosensor, an experiment using 18-baseoligonucleotide sequences of adenine (poly-A) hybridizing to immobilized18-base oligonucleotide sequences of thymine (poly-T) was performedusing a bottomless 96-well plate that was bonded to a biosensor sheet.The poly-A sequence has a Cy-5 labeled attached to its 3′-terminal endto permit a validation of the binding using fluorescence.

Specifically, a biosensor with hydrophobic TaO top layer was firstcoated with poly-phenylalanine-lysine (PPL) (Sigma); the biosensor wasbonded to bottomless polystyrene 96-well microtiter plates (Greiner). Ahybridization buffer solution comprised of 3×SSC buffer (Sigma) and 0.1%SDS (Sigma) was created. Subsequently, 9 wells (three sets of threewells) were utilized to examine three different analytes, each intriplicate. Specifically, in the first and second sets of wells, 10 mMof poly-T (Oligos Etc., Inc.) in water was added. In the third set ofwells, DNA from the T7 promoter region (New England BioLabs) wasimmobilized. After immobilization, hybridization buffer was first usedto rinse all wells, afterwards, the hybridization buffer was added tothe first set of wells, providing a baseline response curve. In thesecond set of wells, poly-A (with Cy-5 label) was added so as to inducehybridization between the immobilized poly-T and the poly-A. In thethird set of wells, poly-A (with Cy-5) was added to the immobilized T7DNA to generate non-specific binding.

FIG. 36 summarizes the results of these experiments. In FIG. 36A, agraphical plot of the amount of binding resulting from poly-T, poly-A,and T7-promoter are shown. This same data is plotted as endpoints, alongwith error bars, in FIG. 36B. It is readily apparent from these plotsthat the biosensor can measure the specific hybridization between poly-Tand poly-A, as well as distinguish such strong interactions from weakernon-specific interactions.

EXAMPLE 17

Protein-DNA Interaction Demonstration on the Colorimetric ResonantReflectance Optical Biosensor

In yet another experiment, the ability to utilize the colorimetricresonant reflectance optical biosensor to detect interaction betweenproteins and DNA was demonstrated. In this case, the interaction betweenT7 promoter DNA and T7 RNA polymerase was used as an illustration.

Using a TaO-coated biosensor that was bonded to the bottom of abottomless 96-well polystyrene microtiter plate (Greiner),poly-phenylalanine-lysine (PPL) (Sigma) was first used to coat thesensor surface. In particular, 12 wells were coated with PPL—three wellsfor each of four analytes to be examined.

In the first set of wells, PBS buffer at pH=7.4 (Sigma) was added. Inthe second set of wells, reaction buffer for T7 RNA polymerase (NewEngland BioLabs) was added. In the third set of wells, the T7-promoterDNA (New England BioLabs) was immobilized first, followed by theaddition of T7 RNA polymerase in reaction buffer (New England BioLabs).In the fourth set of wells, T7-promoter DNA was immobilized, followed bythe reaction buffer. In principle, the T7 reaction buffer should provideonly non-specific responses, unless both T7 DNA and the T7 RNApolymerase are present. As shown in FIG. 37, this was indeed the case.Specifically, note that at the 175th time step a rinse procedure tookplace in order to remove buffer effects; subsequent to this rinse, itwas apparent that protein-DNA interactions occurred only in the wellscontaining both the T7 promoter DNA and the T7 RNA polymerase.

1. A method of detecting a change in activity of one or more enzymes orbinding partners due to one or more molecules, wherein the enzymes orbinding partners cleave or bind one or more specific binding substances,wherein the one or more specific binding substances are immobilized on asurface of a colorimetric resonant reflectance optical biosensor at oneor more distinct locations, comprising: a. detecting a colorimetricresonant reflectance optical PWV (peak wavelength value) of the one ormore distinct locations; b. applying the one or more molecules to theone or more distinct location; c. applying the one or more enzymes orbinding partners to the one or more distinct locations; d. detecting thecolorimetric resonant reflectance optical PWV of the one or moredistinct locations; e. comparing the PWV from step (a) with the PWV fromstep (d); and wherein a change in activity of one or more enzymes orbinding partners due to one or more molecules is detected.
 2. The methodof claim 1, wherein the colorimetric resonant reflectance opticalbiosensor comprises an internal surface of a vessel selected from thegroup consisting of a microtiter well, microtiter plate, test tube,petri dish and microfluidic channel.
 3. The method of claim 1, whereinthe one or more specific binding substances are selected from the groupconsisting of nucleic acids, peptides, protein solutions, peptidesolutions, single or double stranded DNA solutions, RNA solutions,RNA-DNA hybrid solutions, solutions containing compounds from acombinatorial chemical library, antigen, polyclonal antibody, monoclonalantibody, single chain antibody (scFv), F(ab) fragment, F(ab′)₂fragment, Fv fragment, small organic molecule, cell, virus, bacteria,polymer and biological sample.
 4. The method of claim 3, wherein thepolymer is selected from the group of long chain molecules with multipleactive sites per molecule consisting of hydrogel, dextran, poly-aminoacids and derivatives thereof, including poly-l-lysine, poly-d-lysine,poly-phe-lysine and poly-glu-lysine.
 5. The method of claim 1, whereinthe one or more specific binding substances are immobilized onto thesurface of the biosensor via a nickel group, amine group, aldehydegroup, acid group, alkane group, alkene group, alkyne group, aromaticgroup, alcohol group, ether group, ketone group, ester group, amidegroup, amino acid group, nitro group, nitrile group, carbohydrate group,thiol group, organic phosphate group, lipid group, phospholipid groupand steroid group.
 6. The method of claim 1, wherein the one or morespecific binding substances are arranged in an array of distinctlocations on the surface of the biosensor.
 7. The method of claim 6,wherein a distinct location defines an array spot of about 50–500microns in diameter.
 8. The method of claim 1, wherein the one or morespecific binding substances are immobilized on the surface of thecolorimetric resonant reflectance optical biosensor by a method selectedfrom the group consisting of physical adsorption, chemical binding,electrochemical binding, electrostatic binding, hydrophobic binding andhydrophilic binding.
 9. The method of claim 1, wherein the detectioncomprises: a. immobilizing one or more specific binding substances inone or more distinct locations defining an array within a well of amicrotiter plate, wherein the distinct locations defining an array arelocated upon the surface of a colorimetric resonant reflectance opticalbiosensor which comprises an internal surface of the well; b. detectinga colorimetric resonant reflectance optical PWV for the one or moredistinct locations within the well; c. applying the one or moremolecules to the well; d. applying one or more enzymes or bindingpartners to the well; e. detecting a colorimetric resonant reflectanceoptical PWV for the one or more distinct locations within the well; andf. comparing the PWV from step (b) with the PWV from step (e); andwherein a change in activity of one or more enzymes or binding partnersdue to one or more molecules at each distinct location within a well isdetected.
 10. The method of claim 1, wherein the one or more enzymes areselected from the group consisting of proteases, lipases, nucleases,lyases, peptidases, hydrolases, ligases, kinases and phosphatases.